Polyspecific binding molecules and uses thereof

ABSTRACT

The present invention relates to polyspecific binding molecules and particularly single-chain polyspecific binding molecules that include at least one single-chain T-cell receptor (sc-TCR) covalently linked through a peptide linker sequence to at least one single-chain antibody (sc-Ab). Further disclosed are methods and compositions for testing and using the molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 60/105,164 filed on Oct. 21, 1998, the disclosure of which is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to polyspecific binding molecules, as wellas methods of making and using such molecules. In one aspect, theinvention features single-chain polyspecific binding molecules that candamage or destroy target cells. The invention is useful for a variety ofapplications including use in associating cells that express a T-cellreceptor or an antibody binding domain.

BACKGROUND

There has been recognition that immune system cells and particularlycytotoxic T lymphocytes (CTLs) can be used to detect tumor associatedantigens (TAAs). For example, CTLs derived from melanomas have been usedto identify a variety of melanoma-specific antigens. See e.g., Bruggenet al., Science, (1991), 254:1643; Bakker et al., J. Exp. Med., (1994),179: 1005; and Yanuck et al., Cancer Research, (1993), 53, 3257.

Several anti-tumor therapies have attempted to use CTLs to treatdiseases such as cancer. In one approach, anti-tumor CTLs are taken froma patient, expanded in vitro, and then given back to the patient totreat the cancer. However, this approach suffers from significantdrawbacks. For example, it is not always straightforward to isolatesufficient quantities of the CTLs from the patient. In addition, atleast some of the CTLs may have specificities that have survivedself-tolerance that could lead to additional complications. See, e.g.,Browning et al., Curr. Opin. Immunol., (1992) 4, 613; Mizoguchi et al.,Science, (1992), 258:1795, and George et al., J. Immunol., (1994), 152,1802.

There have been attempts to mitigate these and other shortcomings bymaking and using recombinant immune molecules such as those resemblingantibodies. An antibody has a recognized structure that includes animmunoglobulin heavy and light chain. The heavy and light chains includean N-terminal variable region (V) and a C-terminal constant region (C).The heavy chain variable region is often referred to as “V_(H)” and thelight chain variable region is referred to as “V_(L)”. The V_(H) andV_(L) chains form a binding pocket that has been referred to as F(v).See generally Davis Ann. Rev. of Immunology (1985), 3: 537; andFundamental Immunology 3rd Ed., W. Paul Ed. Raven Press LTD. New York(1993).

Recombinant antibody molecules have been disclosed. For example, severalrecombinant bispecific antibody (bsFv) molecules have been reported.Most of the bsFv molecules include a F(v) formatted as a single-chain(sc-Fv). More particular sc-Fv molecules include a V_(H) linked to aV_(I), through a peptide linker sequence. See e.g., Huston et al. PNAS(USA), (1988), 85:5879; Bird et al., Science, (1988), 242: 423; WO94/29350; and U.S. Pat. No. 5,455,030.

Additional bsFv molecules have been disclosed. For example, some bsFvmolecules have been reported to bind a T-cell protein termed “CD3” and aTAA. There is recognition that binding of the bsFv may facilitate animmune system response. See e.g., Jost, C. R. (1996) Mol. Immunol. 33:211; Lindhofer, H. et al. (1996) Blood, 88: 4651; Chapoval, A. I. et al.(1995) J. of Hematotherapy, 4: 571.

There have been attempts to develop straightforward methods of makingbispecific antibody molecules. However, many of these attempts have beenassociated with problems. For example, many of the molecules arereported to be insoluble especially in bacterial expression systems. Seee.g., Wels et al., (1992), Biotechnology, 10:1128.

Attempts to make other recombinant immune molecules have been reported.For example, there have been specific attempts to manipulate T-cellreceptors (TCRs). The TCR is a membrane bound heterodimer consisting ofan α and β chain that resembles an immunoglobulin variable (V) andconstant (C) region. The TCR α chain includes a covalently linked V-αand C-α chain. The TCR β chain includes a V-β chain covalently linked toa C-β chain. See generally Davis, supra.

There have been specific efforts to manipulate the TCR by recombinantDNA techniques. For example, in one approach, the TCR has been formattedas a single-chain fusion protein comprising the TCR V regions (sc-TCR).The sc-TCR molecule has been reported to have several important uses.See e.g., Soo Hoo, W. F. et al. PNAS (USA) 89, 4759 (1992); Wülfing, C.and Plückthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS(USA) 90 3830 (1993); PCT WO 96/13593; PCT WO 96/18105; and Schlueter,C. J. et al. J. Mol. Biol. 256, 859 (1996).

The prior recombinant immune molecules are believed to be associatedwith significant shortcomings.

For example, there has been recognition that many tumor antigens are“shed” from cells, thereby providing sites for non-specific immunemolecule binding. In particular, it has been proposed that many bsFvmolecules inadvertently interact with the shed antigens, therebyreducing tumor cell killing efficiency.

The prior immune molecules suffer from additional drawbacks. Forexample, there has been recognition that many bsFv molecules cannot bindpotential target antigens such as certain peptides on the surface oftumor cells. As an illustration, the tumor related protein p53 isusually not expressed on tumor cells as an intact protein. Instead, p53has been reported to be processed and presented as a peptide in thecontext of a cell surface class I or class II molecule. Thus, insettings in which binding to specific cell surface peptides is needed,it has been difficult or impossible for bsFv molecules.

Further, it has been difficult to isolate some bsFv molecules withoutsignificant isolation and/or re-folding steps. See e.g., Jost, C. R. etal. supra and references cited therein.

Preparation and use of many sc-TCRs has also been associated withproblems. For example, several prior methods for making the sc-TCRs haveyielded insoluble and improperly folded molecules. Several strategieshave been developed in an attempt to improve sc-TCR yields. However, thesc-TCRs produced by these methods often require time-consumingmanipulations to obtain even modest amounts of protein. See e.g., Ward,E. S. et al. supra; Schlueter, C. J. supra; and published PCTapplications WO 96/18105 and WO 96/13593.

There is a need therefore for recombinant immune molecules andparticularly single-chain polyspecific binding molecules that can damageor eliminate (kill) target cells in vitro and in vivo. It would bedesirable to have methods for making the polyspecific binding moleculeswith a minimum of difficult preparative steps.

SUMMARY OF THE INVENTION

The present invention relates to novel immune molecules and particularlyto single-chain polyspecific binding proteins that damage or eliminate(kill) desired target cells. The single-chain polyspecific bindingproteins include at least one receptor domain capable of specificallybinding a peptide bound (loaded) to a major histocompatibility complex(MHC) or a human-leukocyte-associated antigen (HLA); and at least oneantibody domain capable of binding an antigen. The present single-chainpolyspecific binding molecules are fully soluble and can be isolated insignificant quantities with a minimum of difficult preparative steps.Also provided are methods and compositions for screening thesingle-chain polyspecific binding proteins for capacity to bind desiredcells.

We have made novel polyspecific binding molecules that feature a widevariety of useful activities. For example, the single-chain polyspecificbinding proteins can associate cells expressing the peptide bound(loaded) MHC (HLA) to cells expressing the antigen. In most instances,the MHC (HLA) and the antigen will be on separate cells. Association ofthe cells in accord with the invention preferably facilitates an immuneresponse that can damage or kill the cells expressing the peptide bound(loaded) MHC (HLA) complexes. The present invention has a wide spectrumof useful applications including use in the treatment of certain cancersand viral infections.

More particularly, the present invention features single-chainpolyspecific binding proteins that include at least one single-chainT-cell receptor (sc-TCR) or functional fragment thereof sufficient tobind a particular peptide bound (loaded) to the MHC (HLA). A cellexpressing the peptide bound (loaded) MHC or HLA will often be referredto herein as a “target cell” or similar term. The polyspecific bindingproteins further include at least one antibody binding domain andparticularly a single-chain antibody or functional fragment thereof,which antibody binding domain is sufficient to bind the antigen. In mostembodiments, the antigen bound by the antibody binding domain will beexpressed on a cell surface, usually on the surface of an immune cell.In particular embodiments, the antigen will be selective for the immunecells. More preferred single-chain polyspecific binding molecules ofthis invention are capable of forming a specific binding complex(“bridge”) between the peptide bound (loaded) MHC or HLA on the targetcell and the antigen on the immune cell. Without wishing to be bound totheory, it is believed that formation of the bridge in accord with theinvention facilitates an immune response that can damage or kill thetarget cells.

Preferred polyspecific binding molecules of this invention specificallybind MHC or HLA complexes. Unless otherwise specified, the term MHC andHLA as used herein means a complex to which a particular peptide isbound (loaded). In some instances, the MHC (HLA) complexes will bereferenced as “pMHC”, “pHLA” or like term to denote the peptide binding(loading). The polyspecific binding molecules are thus useful forbinding the MHC and HLA complexes and for bridging those complexes to animmune cell expressing a desired antigen. In some instances, the immunecell antigen bound by a particular polyspecific binding molecule will bereferred to as an “activation” molecule or marker to denote preferredactivation of the immune cell following binding by the polyspecificmolecule.

Accordingly, in one aspect, the present invention features single-chainpolyspecific binding proteins that include at least one sc-TCRcovalently linked (i.e. fused) to at least one single-chain antibody(sc-Ab). In embodiments in which the single-chain polyspecific moleculeinclude one sc-TCR and one sc-Ab, the sc-TCR and the sc-Ab molecules maybe directly fused together although it is generally preferred toseparate the sc-TCR and sc-Ab from each other through a suitable (first)peptide linker sequence. Alternatively, functional fragments of thesc-TCR and/or sc-Ab molecules may be employed in the proteins. Inpreferred embodiments, the polyspecific binding proteins will includethe sc-TCR linked to the sc-Ab through the first peptide linkersequence.

More particularly, the sc-TCR is preferably a single-chain V chain. TheV chain will typically include a Vα,β sequence in which a V-α chain isfused to a V-β chain. In a specific embodiment, the fusion is achievedby covalently linking the molecules through a (second) peptide linkersequence. The fusion product may be further covalently linked throughthe V-α or V-β chain to an immunoglobulin constant chain (Ig-C_(L)) orfragment thereof if desired.

In a more specific embodiment, the C-terminus of the sc-TCR V-α chain iscovalently linked by the second peptide linker sequence to theN-terminus of V-β chain. Alternatively, the C-terminus of the sc-TCR V-βchain can be covalently linked by the second peptide linker sequence tothe N-terminus of the V-α chain.

In another embodiment, a TCR C-β chain or fragment thereof is covalentlylinked between the C-terminus of the sc-TCR V-β chain and the N-terminusof the first peptide linker sequence. Alternatively, the TCR C-β chainor the fragment may be covalently linked between the C-terminus of thesc-TCR V-α chain and the N-terminus of the first peptide linkersequence.

In another embodiment, a TCR C-α chain or fragment thereof is covalentlylinked between the C-terminus of the sc-TCR V-α chain and the N-terminusof the second peptide linker sequence fused to the V-β sequence.Alternatively, the C-α chain or fragment can be covalently linkedbetween the C-terminus of the V-β chain and the N-terminus of the secondpeptide linker sequence fused to the V-α sequence.

In a particular embodiment, the sc-TCR includes the TCR C-α chain orfragment covalently linked between the C-terminus of the sc-TCR V-αchain and the N-terminus of the second peptide linker sequence fused tothe sc-TCR V-β sequence. Further, the TCR C-β chain or fragment iscovalently linked between the C-terminus of the V-β chain and theN-terminus of the first peptide linker sequence.

As discussed, the polyspecific binding molecules of this inventioninclude at least one sc-Ab. In a particular embodiment the antibodybinding domain includes at least one sc-Fv. More preferred single-chainpolyspecific binding proteins include one sc-Fv or a functional fragmentthereof. An illustrative sc-Fv includes at least two immunoglobulinchains and especially two immunoglobulin variable chains, e.g., a lightchain (V_(L)) fused to a heavy chain (V_(H)). In this embodiment, theV_(L) and V_(H) chains may be fused together although it is generallypreferred to covalently link the chains through a (third) peptide linkersequence.

In a particular embodiment, the C-terminus of the V_(L) chain iscovalently linked by the third peptide linker sequence to the N-terminusof V_(H) chain. In another embodiment, the C-terminus of the V_(H) chainis covalently linked by the third peptide linker sequence to theN-terminus of the V_(L) chain.

In a more particular embodiment, the C-terminus of the sc-TCR V-β chainis covalently linked to the third polypeptide linker sequence whichsequence is further linked to the N-terminus of the V_(H) chain.Alternatively, the C-terminus of the sc-TCR V-β is covalently linked tothe third polypeptide linker sequence as discussed except that thepolypeptide sequence is further linked to the N-terminus of the V_(L)chain. In another embodiment, the C-terminus of the sc-TCR V-β chain iscovalently linked to a C-β chain which chain is covalently linked to thethird polypeptide linker sequence which sequence is linked to theN-terminus of the V_(H) chain. Alternatively, the C-terminus of thesc-TCR V-β chain is covalently linked to a C-β chain which chain iscovalently linked to the third polypeptide linker sequence whichsequence is linked to the N-terminus of the V_(L) chain.

In a preferred embodiment, the present invention provides single-chain“bispecific” binding proteins that include at least one sc-TCR (orfragment thereof), and at least one sc-Fv (or fragment thereof)covalently linked together through a suitable peptide linker sequence.In instances in which more than one sc-TCR and/or sc-Fv are used thesc-TCRs and sc-Fvs are preferably the same. The single-chain bispecificbinding protein will sometimes be referred to herein as a “bispecifichybrid molecule” or “sc-TCR/scFv hybrid molecule” or similar phrase. Thebispecific hybrid molecules of this invention may include additionalamino acid sequences such as protein tags. More preferred bispecificbinding proteins are discussed as follows.

For example, in one embodiment, the bispecific binding molecules includecovalently linked in sequence: 1) a sc-TCR or functional fragmentthereof of interest, 2) a suitable peptide linker sequence, and 3) asc-Fv or functional fragment thereof. In a more particular embodiment,the sc-TCR further includes covalently linked in sequence: 4) the V-αchain, 5) a suitable peptide linker sequence, 6) a V-β chain, and 7) anoptional C-β chain fragment. Alternatively, the sc-TCR can includecovalently linked in sequence: 4) the V-β chain, 5) the linker sequence,6) the V-α chain, and 7) an optional C-β chain or fragment thereof. Inanother particular embodiment, the sc-TCR further includes a C-α chainor fragment thereof covalently linked between the V-α chain and thepeptide linker sequence fused to V-β chain.

In another particular embodiment, the bispecific binding moleculesinclude a sc-Fv that which includes covalently linked in sequence: 8)the V_(H) chain, 9) a suitable polypeptide linker sequence, and 10) theV_(L) chain. In another embodiment, the sc-Fv includes covalently linkedin sequence: 8) the V_(L) chain, 9) the polypeptide linker sequence, and10) the V_(H) chain.

The single-chain polyspecific binding proteins of this invention mayfurther include at least one protein tag covalently linked thereto,preferably from between about 1 to 3 of such tags. Preferably, theprotein tag is fused to the C-terminus of a desired binding moleculealthough for some applications fusion to the N-terminus may be morepreferred.

In a more specific embodiment, the single-chain polyspecific bindingprotein includes covalently linked in sequence: 1) the TCR V-α chain, 2)a peptide linker sequence, 3) the TCR V-β chain covalently linked to aC-β chain fragment, 4) a peptide linker sequence, 5) the V_(L) chain, 5)a peptide linker sequence, and 6) the V_(H) chain. In anotherembodiment, the single-chain polyspecific binding protein includescovalently linked in sequence: 1) the TCR V-α chain, 2) a peptide linkersequence, 3) the TCR V-β chain covalently linked to a C-β chainfragment, 4) a peptide linker sequence, 5) the V_(H) chain, 5) apolypeptide linker sequence, and the 6) V_(L) chain.

Significantly, the present invention is flexible. That is, the inventionfeatures polyspecific binding molecules that can include a variety ofsc-TCR and sv-FV components. As will be appreciated, the order in whichthe components are made or assembled is usually not important so long asdesired binding and activation characteristics are achieved.

In another embodiment, the present invention features multi-chainpolyspecific binding proteins that include at least one sc-TCR(functional fragment thereof) and at least one antibody binding domainwhich can be, e.g., an F(v) or sc-Fv. The binding molecules morespecifically include at least one “joining molecule” to link the sc-TCRand antibody binding domain. As will be more fully discussed below, thejoining molecule may be covalently or non-covalently linked to thesc-TCR, the antibody binding domain, or both. For example, in onepreferred embodiment, two compatible joining molecules are eachindependently fused to the sc-TCR and the sc-Fv.

The term “joining molecule” means an amino acid sequence that is capableof specifically binding, either covalently or non-covalently, to asecond amino acid sequence. Sometimes the second amino acid sequence isreferred to as a “cognate” sequence to denote capacity to form aspecific binding pair. The second sequence may also be sometimesreferred to herein as a second joining molecule, which second joiningmolecule can be the same as, or different from, the (first) joiningmolecule. More particular joining molecules include immunoglobulinchains and particularly constant chains (H or L) or suitable fragmentsthereof, coiled-coil motifs and helix-turn-helix motifs. More specificexamples of joining molecules are disclosed below.

It will be apparent from the discussion which follows that in someinstances a joining molecule may also serve as a protein tag.

A more particular multi-chain polyspecific binding molecule includesmore than one joining molecule and preferably about 2 of such joiningmolecules. In a more specific embodiment, one sc-TCR is fused to thefirst joining molecule. The first joining molecule can be eithercovalently or non-covalently linked to the second joining molecule whichis further linked to the antibody binding domain. However in someembodiments such as when the first and second joining molecules aresuitable immunoglobulin chains, a combination of covalent andnon-covalent bonds may be employed to link the sc-TCR and the antibodybinding domain through the first and second joining molecules.

As an illustration, a particular multi-chain polyspecific bindingprotein includes covalently linked to at least one sc-TCR, preferablyone sc-TCR, an immunoglobulin heavy chain (Ig-CH) or functionalfragment. Sometimes this construct will be referred to herein as a“sc-TCR/Ig fusion protein”, “sc-TCR/Ig” or similar phrase. It will beappreciated that the immunoglobulin heavy chain portion of the sc-TCR/Igfusion protein is representative of one type of joining molecule asdefined above and in the discussion following. In a more specificembodiment, the binding molecule further includes a second joiningmolecule, which is preferably a suitable immunoglobulin heavy chaincapable of forming a binding complex. The isotype of the immunoglobulinchains may be different but are preferably the same to facilitatebinding. The second joining molecule is bound to the antibody bindingdomain which is preferably an F(v) and particularly an sc-Fv. In otherembodiments, the sc-TCR may be further bound covalently ornon-covalently to an immunoglobulin variable chain and preferably avariable light chain.

The single- and multi-chain polyspecific binding proteins disclosedherein preferably include TCR V-α and the V-β chains that are at least90% identical to T-cell receptor V chains present on a cytotoxic T cell.Preferably, at the least the sc-TCR portion of the protein has beenhumanized and more preferably the entire binding protein has beenhumanized to enhance patient compatibility. In such embodiments it maybe desirable to include at least one protein tag which can be, e.g., adetectably-labeled molecule suitable for diagnostic or imaging studies.

As will be described below, the present polyspecific binding moleculescan be unmodified, or if desired, can be covalently linked to a desiredmolecule, e.g., drugs, toxins, enzymes or radioactive substances througha linked peptide linker sequence.

The polyspecific binding molecules of the present invention provideseveral significant advantages.

For example, preferred, polyspecific binding proteins are capable ofassociating an MHC-expressing target cell and an immune cell. That is,the present binding proteins preferably form a bridge that joins theimmune cell to the MHC- or HLA-expressing cell. As noted, thatassociation is believed to enhance recognition and facilitate damage toor killing of the target cell. In contrast, most prior immune systemmolecules and particularly bsFv molecules are not optimized to bind pMHCor pHLA complexes. Accordingly, the present molecules provide aneffective means for killing target cells that express a pMHC or pHLAmolecule.

Additionally, use of the present polyspecific binding proteins is.believed to be associated with fewer adverse activities when compared tomany prior immune molecules. As an illustration, many prior bsFvmolecules have been reported to bind to shed TAAs. In contrast,preferred polyspecific binding molecules of this invention specificallybind TAAs in the context of MHC or HLA molecules, thereby substantiallyreducing or totally eliminating non-specific binding to the shed debris.Significantly, there has been much less concern in the field regardingany MHC and HLA shedding.

Further, the polyspecific binding molecules disclosed herein can bind asignificantly wider spectrum of molecules than most prior recombinantimmune molecules. In particular, there has been understanding thattargetable antigens are often hidden inside cells making recognition andbinding difficult. It is an object of the present invention to providebinding molecules that specifically bind these hidden antigens. Forexample, the polyspecific binding molecules include at least one sc-TCR(or functional fragment) that can bind antigens in the context of an MHCor HLA. Thus, the present binding molecules are capable of binding alarge variety of antigens that are usually hidden inside cells. Incontrast, most prior recombinant immune system molecules are not able tobind MHC- or HLA-presented antigens effectively.

The present invention provides still further advantages. For example,prior practice generally required extensive manipulation of TCR-relatedproteins (e.g., TCR receptors, TCR heterodimers, sc-TCRs), beforesignificant amounts of protein could be obtained. In contrast, thepolyspecific binding molecules of the present invention are fullysoluble and can be isolated in significant quantities. Additionally, awide variety of the polyspecific binding molecules can be presented forinteraction with various immune system components such as superantigensor APCs.

Additionally, the single- and multi-chain polyspecific binding moleculesinclude immunoglobulin chains that are readily isolated by standardimmunological methods. Presence of these chains can usually facilitatedetection, analysis and isolation of the binding molecules as discussedbelow.

In another aspect, the invention pertains to polynucleotides (RNA, mRNA,cDNA, genomic DNA, or chimeras thereof) that include or consist of asequence that encodes a single- or multi-chain polyspecific bindingprotein. In one embodiment, the polynucleotide includes sequence thatencodes essentially all of the binding protein, e.g., as when thebinding protein is a single-chain construct.

In another embodiment, the polynucleotides include a sequence thatencodes a portion of the polyspecific binding protein and particularlypart of certain multi-chain binding proteins discussed below. Forexample, a particular polynucleotide of this invention may encode ansc-TCR fused to an immunoglobulin heavy chain or suitable fragmentthereof (e.g., an sc-TCR/Ig molecule). In this embodiment, the remainingpart of the polyspecific binding protein can be provided in severalways. For example, it can be provided by a cell or extract thereofcapable of synthesizing antibody molecules such as an antibody bindingdomain. The antibody or antibody-binding domain may be encoded by thecell genome or it may be encoded by an introduced DNA segment.Preferably, the cell will be an antibody-producing cell such as ahybridoma cell. Alternatively, the remaining part of the binding proteinis provided by a second polynucleotide sequence that includes the DNAsegment. In this embodiment, the binding protein is preferablyconstructed by contacting the encoded protein portions together underconditions conducive to forming the desired binding protein. As will bediscussed below, the polyspecific binding proteins of this invention canbe joined by one or a combination of strategies including cellular,genetic and chemical methods.

Particularly contemplated are DNA vectors that include or consist of thepolynucleotides of this invention. Illustrative DNA vectors includethose compatible with conventional prokaryotic or eukaryotic proteinexpression system. More specific examples of polynucleotides and DNAvectors.

The polynucleotides of the present advantage provide importantadvantages. For example, as will become apparent from the disclosurewhich follows, preferred polynucleotides of this invention include DNAsegments that encode covalently linked scTCR and sc-Ab molecules. TheDNA segments are preferably configured in a “cassette” format so that asegment encoding a sc-TCR or sc-Ab can be switched, as desired, withanother segment encoding another scTCR or sc-Ab.

In another aspect, the present invention provides compositions andmethods for selecting polyspecific binding proteins. More particularly,the compositions and methods can be employed to select sc-TCR and sc-Abmolecules with desired characteristics, thereby facilitating manufactureand use of polyspecific binding proteins that include these molecules.

In one embodiment, the invention provides recombinant bacteriophagesthat include at least one sc-TCR (or functional fragment) and at leastone sc-Fv (or functional fragment) as fusion proteins. As will bediscussed, the bacteriophages can be employed, e.g., to select sc-TCRand sc-Fv molecules for desired binding characteristics. Preferred arebispecific bacteriophages. The recombinant bacteriophages may sometimesbe referred to herein as “polyfunctional” or “polyspecific” to denotebinding by the sc-TCR and the sc-Fv fusion proteins. The recombinantbacteriophages can be derived from well-known filamentous “fd” phagesalthough related phages may be used in some cases.

More particularly, the recombinant bacteriophages of this inventioninclude a plurality of fusion proteins that each include: 1) at leastone sc-TCR or functional fragment thereof fused to a first bacteriophagecoat protein, or 2) at least one sc-Ab or functional fragment thereoffused to a second bacteriophage coat protein the same or different fromthe first bacteriophage coat protein. Preferred bacteriophage coatproteins are essentially full-length or may be fragments thereofprovided that the fragment is sufficient to display the fused molecule.By “display” is meant that the protein fusion is part of thebacteriophage coat and is readily detectable on the bacteriophage bystandard screening techniques such as those disclosed below.

In a related aspect, the invention provides a recombinant bacteriophagelibrary that includes a plurality of recombinant bacteriophages in whicheach bacteriophage comprises a plurality of single-chain polyspecificbinding proteins each covalently linked to a bacteriophage coat proteinas a protein fusion, wherein each single-chain binding proteincomprises: 1) one sc-TCR or functional fragment thereof fused to a firstbacteriophage coat protein or fragment, or 2) one sc-Ab or functionalfragment thereof fused to a second bacteriophage coat protein orfragment. More preferred recombinant bacteriophage libraries includebacteriophages that display bispecific binding proteins.

The recombinant bacteriophage libraries can be formatted to include avariety of TCR V chains and/or immunoglobulin variable chains.Accordingly, libraries can be used to select recombinant bacteriophagesthat display desired sc-TCR and sc-Ab molecules.

The recombinant bacteriophages of this invention can be isolated by avariety of conventional techniques. In one embodiment, there is provideda method for isolating the recombinant bacteriophages in which themethods include at least one and preferably all of the following steps:

a) introducing into host cells a first polynucleotide comprising asequence encoding a first fusion protein comprising an sc-TCR covalentlylinked to a first bacteriophage coat protein or fragment,

b) introducing into the host cells a second polynucleotide comprising

a sequence encoding a second fusion protein comprising a sc-Fvcovalently linked to a second bacteriophage coat protein or fragment.

c) culturing the host cells in medium under conditions permittingpropagation of bacteriophages and display of the fusion proteins; and

d) isolating the recombinant bacteriophages from the host cell or themedium.

In a particular embodiment, the method further includes contacting anextract of the host cell or the cultured medium with a synthetic matrixcapable of specifically binding one of the fusion proteins, andpurifying the recombinant bacteriophage from the synthetic matrix toisolate the bacteriophage. In a more particular embodiment, thesynthetic matrix includes an antibody fragment that is capable ofspecifically binding the recombinant bacteriophage. More specificbacteriophage isolation techniques are discussed below.

Additionally provided by the invention is a kit comprising the presentrecombinant bacteriophages which kit may also include suitableprokaryotic cells for propagating the bacteriophage and directions forusing the kit. Also provided is a kit that includes the bacteriophagelibrary discussed above.

The recombinant bacteriophages of this invention have additional usesand advantages. For example, the bacteriophages can be used in accordwith standard screening techniques to facilitate analysis of a desiredpolyspecific binding molecule in vitro. More particularly, therecombinant bacteriophages can be used to assess whether a specificsc-TCR or sc-Ab such as a sc-Fv has capacity to recognize, bind and/orkill target cells of interest. Additional advantages include arelatively fast and straightforward procedure for making and testingbispecific sc-TCR/sc-Ab molecules; a short and simple purificationprocess; and an accelerated method for testing large numbers ofdifferent hybrid molecules for efficacy in damaging or killing targetcells (e.g., tumor killing).

The single- and multi-chain polyspecific binding proteins of thisinvention can be made as fully functional and soluble proteins by one ora combination of methods. In general, the methods involve cellular,recombinant DNA and chemical techniques, or combinations thereof.

For example, in one embodiment, there is provided a method for making asingle-chain polyspecific binding protein comprising at least one sc-TCRor functional fragment thereof and at least one sc-Ab and particularly asc-Fv or functional fragment thereof. The method includes at least oneand preferably all of the following steps:

a) introducing into a host cell a DNA vector encoding a single-chainpolyspecific binding protein of interest,

b) culturing the host cell in media under conditions sufficient toexpress the single-chain polyspecific binding protein in the cell or themedia; and

c) isolating the single-chain polyspecific binding protein from the cellor media.

Additionally provided are methods for making a multi-chain polyspecificbinding protein comprising at least one sc-TCR or functional fragmentthereof and an antibody binding domain or functional fragment. In oneembodiment of the method, the antibody-binding domain is a F(v). Inparticular, a cell or cell extract is used to form at least part of themulti-chain binding protein. More particularly, an antibody producingcell such as a hybridoma is employed. In one embodiment, the methodincludes at least one and preferably all of the following steps:

a) introducing into a hybridoma cell a DNA vector encoding at least onesc-TCR, preferably one sc-TCR (or functional fragment) covalently linkedto an immunoglobulin constant heavy chain or fragment thereof,

b) culturing the hybridoma cell in media under conditions conducive toforming a specific binding complex between the immunoglobulin constantheavy chain or fragment encoded by the DNA vector and immunoglobulinchains produced by the hybridoma; and

c) purifying the multi-chain polyspecific binding protein from thehybridoma cells or media.

In a more specific embodiment, the method provides for a multi-chainpolyspecific binding protein that includes an immunoglobulin variablelight chain covalently linked to the sc-TCR, i.e., a bispecific bindingprotein.

The present invention provides additional methods for making themulti-chain polyspecific binding proteins. For example, in oneembodiment, each chain of a desired binding protein is madeindependently, e.g., by recombinant DNA or chemical methods. Preferably,the binding protein further includes at least one joining molecule,preferably two joining molecules the same or different. In a particularembodiment, the method includes at least one and preferably all of thefollowing steps:

a) providing a first sequence that includes at least one sc-TCR orfunctional fragment thereof covalently linked to a first joiningmolecule,

b) contacting the first sequence with a second sequence that includes atleast one sc-Fv or functional fragment thereof linked to a secondjoining molecule, wherein the contacting is under conditions sufficientto form a specific binding complex between the first and second joiningmolecules; and

c) forming the multi-chain polyspecific binding protein.

Preferably, the multi-chain polyspecific binding protein is bispecific.

More specific recombinant DNA and chemical methods for making themulti-chain polyspecific binding proteins are disclosed below.

As discussed, the present polyspecific binding proteins also havesignificant uses in vivo. For example, the binding proteins can be usedto redirect the specificity of certain immune cells, e.g., to eliminatetarget cells such as virally-infected or tumor cells. In some instances,the tumor cells may also be virally-infected. As discussed, preferreduse of the present binding molecules can increase damage or eliminationof the target cells. Accordingly, the present invention can be used invivo to kill target cells by enhancing immune system activation againstthose target cells. Preferred in vivo use of the present polyspecificbinding molecules includes use in a mammal such as a rodent, primate ordomesticated animal, and especially a human patient.

Thus, in one aspect, the invention provides methods for damaging orpreferably killing a target cell comprising an MHC or HLA of interest.In one embodiment, the method includes at least one and preferably allof the following steps:

a) contacting a plurality of cells with a polyspecific binding protein,wherein the plurality of cells comprises immune cells comprising anantigen and target cells comprising the MHC or HLA,

b) forming a specific binding complex (bridge) between the MHC or HLA onthe target cells and the antigen on the immune cells sufficient toactivate the immune cells; and

c) killing the target cells with the activated immune cells. It will beappreciated that by the term “activated” is meant that the immune cellsare capable of damaging or killing the target cell as determined, e.g.,by cytokine and cytotoxicity assays described below.

If desired, the above-described method may be conducted in vitro such asin a cell culture dish.

The single- and multi-chain polyspecific binding proteins of thisinvention have additional uses in vitro and in vivo.

For example, preferred polyspecific binding molecules of this inventioncan be used in vitro or in vivo to detect and preferably form a bridgebetween target cells and immune cells. Formation of the bridge can beused to isolate cells expressing desired MHC, HLA or antigen markers.

The present polyspecific binding proteins also find use in the detectionand analysis of MHC or HLA molecules and cell surface antigens. Thepresent binding proteins can also be used for diagnostic purposes suchas for the detection of immune system cells and especially T-cells withpathogenic properties. The present binding molecules can additionally beemployed in functional, cellular and molecular assays, and in structuralanalysis, including X-ray crystallography, nuclear magnetic resonanceimaging, computational techniques such as computer graphic display.

In another aspect, the present invention further provides treatmentmethods for reducing or eliminating presence of the target cells in amammal. In particular, the methods include administering a polyspecificbinding protein of this invention in a pharmaceutically acceptableformulation. If desired, the sc-TCR or sc-Ab portion of particularpolyspecific binding molecule can be removed prior to or during theadministration to facilitate a specific treatment method.

In a more particular embodiment, the treatment methods are employed totreat cancer and a viral infection. In particular, the methods includeadministering a therapeutically effective amount of at least onepolyspecific binding protein of this invention to a mammal andespecially a human patient. Preferably the amount is sufficient to treatthe cancer and/or the viral infection. The methods may be used to treatan existing condition or may be used prophylactically as needed. Thepresent treatment methods may be used alone or in combination with othertherapies if desired.

Preferred treatment methods of the invention reduce or eliminatepresence of specific target cells in a mammal, particularly a rodent ora primate such as a human. In one embodiment, the treatment methodsinclude obtaining an sc-TCR or sc-Ab from the polyspecific bindingmolecule (e.g., by protease treatment). The sc-TCR or sc-Ab so obtainedmay be administered to the mammal instead of or in conjunction with thepolyspecific binding molecule. For most applications involving animaluse, it will be preferred to minimize undesired immune responses againstthe present binding molecules, e.g., by using immunoglobulin chains of ahaplotype compatible with the animal being used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D are drawings showing single-chain T-cell receptor (sc-TCR)and single-chain Fv (sc-Fv) DNA constructs. (1A) D011.10 sc-TCRconstruct, (1B) p149 sc-TCR construct, (1C) 145-2C11 sc-Fv, (1D) F23.1scFv DNA construct.

FIGS. 2A-2E are drawings showing sc-TCR inserts of various vectors: (2A)pSun22; (2B) pSun23; (2C) pSun21; (2D) pSun19; and (2E) pSun20.

FIG. 3 is a schematic illustration of the pSUN23 vector.

FIG. 4 is a schematic illustration of the pNAG2 vector.

FIG. 5 is a schematic drawing showing the pSUN27 vector.

FIG. 6 is a drawing showing preferred bispecific hybrid moleculespBISP/D011.10 and pBISP/149.

FIG. 7A is a schematic drawing showing a method for making a chimericbispecific antibody molecule. The method uses a hybridoma-expressingcell (145-2C11 hybridoma) to produce antibody chains (heavy lines) thatcombine with an sc-TCR/Ig fusion molecule (light chain) inside the cell.A preferred structure for the sc-TCR/Ig molecule is illustrated in FIG.7B.

FIG. 8 is a drawing showing the vector pSUN7 vector.

FIGS. 9A-9B are graphs showing results of ELISA assays used to detectBISP/149 fusion protein. Antibodies used were (9A) αEE (capture Ab) andαV_(α)2 (B20.1, probe Ab), (9B) αVβ11 (RR3-15, capture Ab). RR3-15 is amonoclonal antibody specific for the αVβ11 chain. B20.1 is an monoclonalantibody specific for the αVα2 chain. Probe Ab is on the x-axis in FIG.9B.

FIGS. 10A-10B are graphs showing results of ELISA assays used to detectBISP/D011.10 fusion protein. Antibodies used were (10A) F23.1 (probe Ab)or (10B) H57-597 (probe Ab). Capture Ab is on the x-axis in FIGS. 9A and9B.

FIG. 11 is a graph showing results of ELISA assays used to detectchimeric bispecific molecules. Capture Ab is goat anti-mouse-IgG2b(x-axis). Probe Ab is goat anti-hamster IgG.

FIG. 12 is a representation of a Western Blot showing expression ofBISP/D011.10 and BISP/p149 bispecific hybrid molecules. Also shown arereduced forms of the proteins (arrow).

FIG. 13 is a representation of an SDS gel stained with Coomassie-blue.The gel shows expression of BISP/D011.10 and BISP/p149 bispecific hybridmolecules. Also shown are reduced forms of the proteins (arrow).

FIG. 14 is a graph showing IL-2 levels expressed by T-hybridoma cells asa function KJ-1 monoclonal antibody.

FIG. 15 is a graph showing IL-2 expressed by T-hybridoma cells as afunction BISP or BPSP plus MR-5 antibody addition.

FIG. 16 is a graph showing IL-2 expression of T-hybridoma cells as afunction of monoclonal antibody addition.

FIG. 17 is a graph showing by flow cytometry binding of pBisp 149 cellsto 2B4 T cells.

FIG. 18 is a graph showing by flow cytometry binding of thepBISP/D011.10 purified protein between 2B4 cells T-cells.

FIG. 19 is a graph showing flow cytometry binding studies betweensoluble α-CD3 and pBISP/149 purified protein.

FIG. 20 is a graph showing results of a T-cell proliferation assay.BALB/c splenocytes were pre-stimulated with rIL-2 and incubated in theabsence or presence of 10 μg/well of enriched scBisp 149 molecule withunpulsed target cells (T2) or p149 peptide pulsed T2 cells.

FIGS. 21A-21B are drawings showing specific oligonucleotide primers (SEQID NOs: 16-43).

DETAILED DESCRIPTION OF THE INVENTION

As summarized above, the present invention features, in one aspect,single-chain polyspecific binding proteins and methods for making andusing the proteins. Preferred use of the present binding moleculesincludes damaging or eliminating (killing) MHC-expressing target cellsin vitro or in vivo. Further provided are highly useful recombinantbacteriophages and methods of using same that can be used to select fordesired binding molecules.

As used herein, the term “polyspecific binding protein” or similarphrase means a single-chain or multi-chain molecule that preferablyincludes 1) a binding domain capable of binding an MHC or HLA complex,preferably a cell target expressing an pMHC or pHLA (or portion thereof)and 2) an antibody binding domain capable of binding an antigen target,preferably an antigen or epitope portion thereof expressed on thesurface of an immune cell. Preferably the pMHC or pHLA portion iscapable of being specifically bound by a TCR and the antigen portion iscapable of being specifically bound by an antibody. In preferredembodiments, the antigen is a cell surface antigen that is indicative ofthe immune cell. In additionally preferred embodiments, each of thebinding domains is sufficient to bind the pMHC or pHLA, and antigentargets at alternate times or at the same time. As discussed herein, thebinding domains may be present on the same chain (i.e. on asingle-chain) or the binding domains may be present on more than onechain (i.e. on a multi-chain and particularly from between about 2 to 4chains with 2 chains being preferred.)

By the term “antibody binding domain” is meant an antibody binding sitecomprising at least one and preferably two immunoglobulin variablechains that are capable of specifically binding the antigen or epitopethereof. For example, in a preferred embodiment, the antibody bindingdomain is a single-chain construct (sc-Ab) and includes a singleimmunoglobulin variable region (V_(L) or V_(H)); two or more variableregions (V_(L)+V_(H); V_(L)+V_(L); or V_(H)+V_(H)); or the complementarydetermining regions thereof. A more particular example of a suitablesc-Ab antibody binding domain is a sc-Fv molecule. In anotherembodiment, the antibody binding domain further includes animmunoglobulin constant light chain (Ig-C_(L)) and/or an immunoglobulinheavy chain (Ig-C_(H)). Preferred examples include, but are not limitedto, Fab, F(v), Fab′ and F(ab′)2 molecules. More specific antibodybinding domains are discussed below.

In a more particular embodiment, the polyspecific binding protein is asingle-chain construct that includes at least one sc-TCR (or functionalfragment) and at least one sc-Ab and especially a sc-Fv (or functionalfragment). The single-chain polyspecific binding protein can includefrom between about 1 to 5 sc-TCR molecules and/or from between about 1to 5 sc-Fv molecules with one sc-TCR and one sc-Fv being generallypreferred for most applications. In embodiments in which the bindingprotein includes at least one sc-TCR or sc-Fv, the molecules may belinked in tandem and are preferably separated from each other bysuitable peptide linker sequences.

More particular polyspecific binding molecules of this invention arebispecific binding molecules and include one sc-TCR and one sc-Ab andparticularly one sc-Fv molecule. In this embodiment, the sc-TCR and thesc-Fv are separated by a suitable peptide linker sequence.

In general, the present polyspecific binding proteins includepre-determined binding specificities. That is, choice of a particularsc-TCR or antibody binding domain will be guided by recognizedparameters such as intended use and the target cells and immune cells ofinterest. In most instances, the binding specificities will be differentas determined by specific binding assays described below. However, insome embodiments, it will be useful to select binding domains with thesame or closely related binding specificities. Methods for selectingdesired binding domains and for choosing appropriate sc-TCR and sc-Abmolecules are described below.

As discussed, in one embodiment, the present polyspecific bindingproteins include an scTCR or functional fragment thereof that bindspMHC- or pHLA-expressing target cells. In a more particular embodiment,the scTCR is chosen to specifically bind a class I or class II pMHCmolecule (or an pHLA antigen) on the target cell. More specificdisclosure relating to sc-TCR molecules including methods for making andusing same have been disclosed in the pending U.S. application Ser. No.08/813,781, filed on-Mar. 7, 1997 and 08/943,086, filed on Oct. 2, 1997.The pending U.S. application Ser. Nos. 08/813,781 and 08/943,086 areincorporated herein by reference.

As used herein, the term sc-TCR “functional fragment” means a portion ofa full-length sc-TCR (i.e., comprising full-length V chains) that iscapable of specifically binding at least about 70% and preferably atleast about 80%, 90%, 95% up to 100% or more of an MHC or HLA whencompared to a full-length sc-TCR. A full-length sc-TCR is defined as amolecule with a full-length V-α and V-β chain. Assays for detectingspecific binding are discussed below and include flow cytometry andBiaCore.

As mentioned previously, the sc-TCR includes TCR V-α and V-β chainscovalently linked through a suitable peptide linker sequence. Forexample, the V-α chain can be covalently linked to the V-β chain througha suitable peptide linker sequence fused to the C-terminus of the V-αchain and the N-terminus of the V-β chain. The V-α and V-β chains of thesc-TCR fusion protein are encoded by nucleic acids generally about 200to 400 nucleotides in length, preferably about 300 to 350 nucleotides inlength, and will be at least 90% identical, and preferably 100%identical to the V-α and V-β chains of a naturally-occurring TCR. By theterm “identical” is meant that the amino acids of the V-α or V-β chainare 100% homologous to the corresponding naturally-occurring TCR V-β orV-α chains. See Examples 1-3 below and the pending U.S. application Ser.Nos. 08/813,081 and 08/943,086 for more specific disclosure relating tosc-TCR V chains.

As mentioned previously, the V-α chain of the sc-TCR molecule canfurther include a TCR C-β chain or fragment thereof fused to theC-terminus of the V-β chain. Further, the V-α chain can include a TCRC-α chain or fragment thereof fused to the C-terminus of the V-α chainand the N-terminus of the peptide linker sequence. Generally, in thosesc-TCR fusion proteins including a C-β chain fragment, the fragment willhave a length of approximately 50 to 126 amino acids and will usuallynot include the last cysteine residue at position 127. For those fusionproteins comprising a C-α chain, the length can vary betweenapproximately 1 to 90 amino acids (i.e. the C-α chain up to but notincluding the final cysteine). For example, in one embodiment, thefusion protein includes a C-α chain fragment between about 1 to 72 aminoacids starting from amino acid 1 to 72. In another embodiment, the C-αchain fragment is between about 1 to 22 amino acids starting from thefirst amino acid to 22 (leucine). The C-α chain fragment typically doesnot include any cysteine resides except the C_(∝90) variant whichincludes two cys residues. In most cases, choice of Cα and Cβ chainlength will be guided by several parameters including the particular Vchains selected and intended use of the particular polyspecific bindingprotein. See the following discussion and examples 1-2 below for morespecific disclosure relating to sc-TCR C-β and C-α chains. See also thepending U.S. application Ser. Nos. 08/813,781 and 08/943,086.

As disclosed in the pending U.S. application Ser. No. 08/943,086, it ispossible to facilitate expression of fully soluble and functional sc-TCRfusion proteins by adding an Ig-C_(L) chain or suitable Ig-C_(L)fragment thereof. More specifically, Ig-C_(L) chain or chain fragment iscovalently linked to the sc-TCR molecule, e.g., to the C-terminus of theV-β chain or C-β fragment. Although typically not preferred, it ispossible to covalently link the Ig-C_(L) or fragment thereof to theN-terminus of the V-α chain. If desired, the Ig-C_(L) chain can beremoved prior to incorporation into a polyspecific binding molecule.

As discussed above, the sc-TCR of a polyspecific binding protein may beprovided in a variety of suitable formats. For example, the sc-TCR maybe provided with e.g., two peptide linker sequences, where the firstpeptide linker sequence is fused between the C-terminus of the V-α chainand the N-terminus of the V-β chain. The C-terminus of the V-β chain canbe fused to the N-terminus of a C-β chain fragment if desired. Thesecond peptide linker is then fused to the C-terminus of the V-β chainor C-β chain fragment and the N-terminus of, e.g., an effector orprotein tag.

In other illustrative embodiments, the sc-TCR of the polyspecificbinding molecule includes a V-β chain fused to the V-α chain through asuitable peptide linker in which the C-terminus of the V-β chain or C-βchain fragment thereof and the N-terminus of the V-α chain arecovalently linked.

The aforementioned sc-TCR molecules are further fused to a peptidelinker sequence which sequence is typically further covalently linked toan antibody binding domain of interest. Preferred single-chainpolyspecific binding proteins include covalently linked in sequence ansc-TCR, a peptide linker sequence, and a sc-Ab such as a sc-Fv.

As disclosed in the pending U.S. application Ser. Nos. 08/813,781 and08/943,086, it is possible to make and use a variety of sc-TCRmolecules. In particular, it is generally preferred that the sc-TCRinclude Vα,β chains for which a full-length or substantially full-lengthcoding sequence is readily available. Methods for obtaining full-lengthTCR V chain sequences from cell sources are well known. Alternatively,the Vα,β chain regions can be obtained by PCR amplication of publiclyavailable Vα,β chains. Exemplary Vβ gene sequences include V β8.1, Vβ6.1, V β5.1, V β5.2, V β5.3, V β2.1, and V β2.3 gene sequences. See Abeet al. (1992) PNAS (USA) 89: 4066; Wang, et al., 1993); PNAS (USA) 90:188; Lahesma et al. (1993) J Immunol. 150: 4125; Kotzin, et al., (1991)PNAS (USA) 88: 9161; Uematsu, et al. (1991) PNAS (USA) 88: 8534. Seealso, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, (5th Ed.) Public Health Science, NIH, and Chotia, C. et al.,(1988) EMBO J. 7:3745 for additional TCR V-β, Vα chain sequences.

In addition, Examples 1-3 below provide oligonucleotide primers for PCRamplifying specific V-α and V-β chains. See also, FIGS. 21A-21B forexamples of oligonucleotide primers that can be used to isolate the TCRV chains.

In cases where it is desired to obtain TCR V chains from a biologicalsource, a desired TCR can be identified by conventional immunologicalmethods including use of TCR-specific antibodies, which predominantlybind, and preferably are specific for, an epitope of the TCR V region.Typically, surface expression can be detected by using known techniquessuch as fluorescence microscopy, flow cytometry, or immunochemistry. Anumber of antibodies which specifically bind TCR variable regions areknown. See e.g., published PCT application WO 90/06758.

The DNA or RNA of the detected TCR can be probed directly, or preferablyafter PCR amplification, by specific hybridization with oligonucleotideprobes for the various TCR gene families, using hybridization methodswell-known in the field. Generally, high stringency nucleic acidhybridization conditions will be performed. As used herein the term“high stringency hybridization” means nucleic acid incubation conditionsapproximately 65° C. in 0.1×SSC. See Sambrook, et al., infra. The TCRDNA sequence or desired portion thereof can be obtained directly fromthe amplified DNA or RNA and can be subcloned into a suitable vector asdesired.

Other methods are known for obtaining TCR V region DNA. For example, adesired TCR comprising V region genes can be identified by sequencingthe TCR or preferably a portion thereof corresponding to the V region.The DNA sequence can be determined, e.g., after cloning DNA into asuitable sequencing vector as are known in the field or by firstdetermining the protein sequence of at least part of the TCR anddetermining the DNA sequence. It will be readily apparent to thoseskilled in this field that the above-mentioned manipulations as well asothers known to the artisan can be employed to successfully identify adesired TCR and to obtain the V region genes from that TCR so that asingle-chain Vαβ construct can be made.

More specifically, when it is desired to obtain TCR V region DNA from abiological source, a DNA segment encoding the desired V-α and V-β chaincan be obtained from cells such as T-cell hybridomas or cytotoxicT-cells (CTLs). The T-cells (e.g., T_(S), T_(C) or T_(H) cells) can beobtained in vivo, or the T-cells can be cultured T-cell hybridoma(s)(e.g., D10 or B12 cell lines). See Examples 1-3 which follows and thepending U.S. application Ser. Nos. 08/813,781 and 08/943,086. CTLs canbe uninduced or can be associated with a pathogenic immune systemresponse in a rodent (e.g., mouse, rat, rabbit) or primate (e.g. humanor chimpanzee). For example, CTLs or other T-cells can be derived frompatients suffering from or suspected of having Lyme disease, Kawasakidisease, leprosy, cancer (i.e. immune responses against tumor associatedantigens such as CEA), or an autoimmune disorder, particularly thoseassociated with transplantation rejection, multiple sclerosis, insulindependent diabetes, rheumatoid arthritis, and allergies; or aninfectious disease, particularly an infectious disease involving an RNAor DNA virus. Particular viruses of interest include the humanimmunodeficiency viruses (HIV), cytomeglovirus (CMV), influenza,hepatitis, pox virus, Epstein Barr, adenovirus or polyoma viruses.Exemplary sources of CTLs are antigen-specific CTLs and TILs isolatedfrom patients with established carcinomas and melanomas (see e.g., CoxA. et al. Science (1994) 264: 716; Rosenberg, S. A. et al. N. Eng. J.Med. (1988) 319: 1676; Kawakami, Y. et al., J. Exp. Med. (1994) 180:347); Kawakami, Y. et al. PNAS (1994) 91:6458).

As mentioned previously with respect to obtaining V-α and V-β chainsfrom cell sources, several alternative procedures can be used to preparenucleic acids isolated therefrom. More particularly, to prepare V-α andV-β chain DNA, mRNA is isolated from those cells demonstrating a desiredTCR binding specificity. Such methods generally include use of asuitable PCR protocol using first-strand cDNA template made from themRNA. Standard recombinant techniques can then be employed to make thedesired α and β chains. The DNA segment encoding the desired V-α and V-βchains is then modified to include a suitable peptide linker sequenceand protein tag(s), if desired.

Generally, a DNA oligonucleotide primer for use in the PCR methods willbe between from about 12 to 50 nucleotides in length preferably frombetween about 20-25 nucleotides in length. The PCR oligonucleotideprimers may suitably include restriction sites to add specificrestriction enzyme cleavage sites to the PCR product as needed, e.g., tointroduce a ligation site. Exemplary primers are provided in theExamples and Drawings which follow. The PCR products produced willinclude amplified V-α and V-β chain sequences and can be modified toinclude, as desired, ribosome binding, leader and promoter sequences foroptimal expression of the fusion protein.

A DNA segment encoding a desired sc-TCR molecule can be made insignificant quantities (milligram quantities per gram cells) in accordwith methods disclosed below and in the pending U.S. application Ser.No. 08/943,086.

More particular sc-TCRs used to make the present polyspecific bindingproteins include those sc-TCRs with V-α and V-β chains derived from amammal. Examples include primates, particularly human and chimpanzees;rodents, e.g., immunologically naive mice such as nude mice or micewhich include a transgene capable of expressing an HLA-A2 antigencomplex (Vitiello, A. et al., J. Exp. Med., (1991) 175, 1002).Particular humans of interest include those suffering from any of thepreviously mentioned pathologies, such as an autoimmune disorder.Chimeric constructs comprising V-α and V-β DNA sequences derived fromdifferent mammals can be constructed in accordance with known methodsand are also within the scope of the present invention.

It is preferred that a peptide linker sequence used to make the sc-TCRbe capable of effectively positioning the V-α and V-β chains to form aligand binding pocket. The sc-TCR is thus preferably capable ofspecifically binding a desired ligand such as a superantigen or peptideantigen in the context of an MHC/HLA peptide complex, or a smallmolecule. In some embodiments of the present invention, the polyspecificbinding molecules may be used to compete with naturally-occurring TCRson the surface of T-cells. By “compete” is meant that the soluble fusionprotein is able to bind the ligand at a level which is equal to, or insome instances exceeds the specific binding affinity of the TCR for thesame ligand. For example, in accordance with methods described below andin the pending U.S. application Ser. No. 08/943,086, the sc-TCR fusionprotein (or sc-TCR molecule derived therefrom) can exhibit a bindingaffinity which is about equal or up to approximately 2 to 10 fold higherthan the naturally-occurring TCR. Exemplary binding assays are disclosedherein and include standard Western blotting assays and surface plasmonresonance assays disclosed below and in the pending U.S. Application.

In general, the peptide linker sequences disclosed herein (sometimesreferred to as a polypeptide linker, spacer sequence, peptide linker orrelated term) are selected to maximize binding interactions between aparticular polyspecific binding molecule and its binding target ortargets. For example, a peptide linker sequence suitable for the sc-TCRis preferably selected so that the sc-TCR forms a specific binding sitewhich resembles that of a naturally occurring TCR V-α and V-β chain.Additional peptide linker sequences such as those used for making sc-Abmolecules are also selected to optimize binding to specific antigens. Insingle-chain constructs, peptide linker sequences fusing the sc-TCR tothe sc-Ab are selected typically maximize interaction between thesc-TCR, the sc-Ab, and respective targets of those binding units.

More particularly, the peptide linker sequence separating the Vα,βchains of the sc-TCR preferably flexibly positions the V-chains in apocket that is capable of specifically binding ligand. Preferred ligandsin this instance are antigens and especially peptide ligands presentedin the context of an MHC. As will be explained more fully below, in thediscussion that follows ligand binding to the sc-TCR can be used tomodulate T-cell activity as determined by specific assays describedbelow. Exemplary of such assays include in vitro assays involvingsequential steps of culturing T-cells expressing a TCR, contacting theT-cells with the sc-TCR protein (or sc-TCR obtained therefrom) underconditions which allow binding between the TCR and the ligand, and thenevaluating whether the soluble fusion protein is capable of modulatingactivity of the T-cells.

In a more specific embodiment, the polypeptide linker sequence comprisesfrom about 7 to 25 amino acids, more preferably from about 10 to 20amino acids, still more preferably from about 12 to 20 amino acids. Thelinker sequence is typically flexibly disposed in the fusion protein soas to position the V-α and V-β chains in a configuration which providesfor specific binding of a desired ligand such as a peptide antigen. Thelinker preferably predominantly comprises amino acids with small sidechains, such as glycine, alanine and serine, to provide optimalflexibility. Preferably, about 80 or 90 percent or greater of the linkersequence comprises glycine, alanine or serine residues, particularlyglycine and serine residues. Preferably, the linker sequence does notcontain any proline residues, which could inhibit flexibility. Thelinker sequence is suitably attached to the C-terminus of the V-α chainand the N-terminus of the V-β chain of a fusion protein. See Examples1-3 and 5 below for disclosure related to making and using specificpeptide linker sequences.

More specifically, suitable peptide linker sequences in accord with theinvention include between from about 5 to 25 amino acid sequences suchas the (GGGGS)₄ sequence (i.e., Gly Gly Gly Gly Ser)₄ (SEQ ID NO: 1).Preferably, a selected peptide linker sequence is covalently linkedbetween the C-terminal residue of the V-α chain, and the first aminoacid of the V-β chain of the sc-TCR. Several polypeptide linkersequences have been disclosed as being acceptable for use in joiningantibody variable regions (see M. Whitlow et al., Methods: A Companionto Methods in Enzymology, 2:97-105 (1991)). Many of those reportedpeptide linker sequences can be used to make the sc-TCR.

Alternatively, other suitable linker sequences can be readily identifiedempirically. For example, a DNA vector including a DNA segment encodinga fusion protein that includes the linker sequence can be cloned andexpressed, and the fusion molecule tested to determine if the moleculeis capable of binding antigen. An exemplary assay is a conventionalantigen binding assay such as those disclosed in Harlow and Lane, supra.Alternatively, the expressed fusion protein comprising the linkersequence can be tested for capacity to modulate the activity of a T-cellas determined by assays disclosed herein. Suitable size and sequences oflinker sequences also can be determined by conventional computermodeling techniques based on the predicted size and shape of the fusionprotein. Exemplary peptide linker sequences are those which includesuitable restriction sites (e.g. XhoI and SpeI) at the ends of thepolypeptide linker sequence between the Vα and V-β chains.

Although the foregoing discussion has focused on selection of suitablesc-TCR peptide linker sequences, it will be understood that similarconsiderations can be used to select other peptide linker sequencesuseful for making the polyspecific binding molecules of this invention.Additional peptide linker sequences include those that are used to makecertain antibody binding domains and particularly the sc-Ab, as well aspeptide linker sequences used to join the sc-Ab to the sc-TCR insingle-chain constructs.

In particular, preferred peptide linkers for making sc-Ab molecules andespecially sc-Fv molecules are usually helical in structure. In general,such peptide linker sequences facilitate proper folding of the sc-Fv andcan enhance the solubility of the sc-Fv and the polyspecific bindingprotein. More preferred peptide linker sequences include from betweenabout 5 to 25 amino acids and preferably from between about 10 to 25amino acids. More specific disclosure relating to suitable sc-Fv peptidelinker sequences can be found in U.S. Pat. No. 5,637,481 to Ledbetter etat the disclosure of which is incorporated by reference.

More preferred sc-Fv peptide linker sequences include the followingpeptide sequences: (G₄S)₃(i.e. Gly Gly Gly Gly Ser)₃ (SEQ ID NO. 2) and(G₄ SG₄A PG₄S) (i.e. Gly Gly Gly Gly Ser Ala Pro Gly Gly Gly Gly Ser)(SEQ ID NO. 3). See FIGS. 1A-B and Examples 4, 5 below.

Preferred peptide linker sequences for joining the sc-TCR to the sc-Fvinclude can be the same or closely related to those peptide linkersequences used to make the sc-TCR. More preferred are peptide linkersequences having the following sequences: (G₄S)₄ (SEQ ID NO. 1) andVNAKTTAPSVYPLEPVSGSSGSG (SEQ. ID NO. 4). See also FIG. 6 and Example 7below.

The sc-TCR of the present binding molecules can be prepared as discussedabove, as well as the examples which follow. See also the pending U.S.application Ser. Nos. 08/813,781 and 08/943,086. Generally, DNA codingfor a desired V-α or V-β chain can be obtained from a suitable sourcesuch as a T-cell, T-cell hybridoma line, or publicly available V-α andV-β chain sequence as described previously. The DNA can be amplified byPCR, cloning or other suitable means. For example, DNA encoding adesired V-α chain can be cloned into a suitable vector, followed bycloning of DNA encoding a desired V-β chain and a suitable single chainlinker sequence to produce a desired sc-TCR. As disclosed previously, insome cases the sc-TCR will include a DNA encoding a C-α and/or C-β chainfragment. In some instances it may be useful to further fuse an Ig-C_(L)chain or fragment to the sc-TCR e.g., the murine or human Cκ chain orsuitable Cκ chain fragment. As noted previously, DNA encoding the Cκchain can be PCR amplified and ligated to DNA encoding the sc-TCR.Alternatively, the Cκ chain can be included in a DNA vector such asthose disclosed by Near, et al., infra. The DNA segment encoding thefusion protein is then introduced into the DNA vector. The DNA vector isthen expressed in a host cell and fusion protein harvested and purifiedif desired.

Illustrative sc-TCRs are generally encoded by a DNA segment includingcovalently linked in sequence: promoter/leader sequence/V-αchain/single-chain linker sequence/V-β chain; promoter/leadersequence/V-α chain/single-chain linker sequence/V-β chain, C-β chainfragment; promoter/leader sequence/V-α chain, C-α chain/single chainlinker sequence/V-β chain/Cκ chain; or promoter/leader sequence/V-αchain, C-α chain fragment/single-chain linker sequence/V-β chain, C-βchain fragment. Additional sc-TCR molecules are as described aboveexcept that a C_(K) chain is fused to the DNA segment. The DNA vectorsencoding the sc-TCR proteins are introduced into desired cells,including those specific expression systems disclosed herein, forsoluble expression of the fusion protein.

As discussed, the single-chain variable regions of the present bindingmolecules can be derived from nearly any suitable TCR or immunoglobulinvariable region. With respect to the TCR portion of the present bindingmolecules, suitable Vα, β chains will be those for which there is anincrease in gene expression following immunological induction. Methodsfor assaying an increase in TCR V chain expression are known (see e.g.,Hafler, D. A. et al. J. Exp. Med. 167: 1313 (1988); and Mantgazza R, etal. Autoimmunity 3, 431 (1990)).

Additionally specific sc-TCR molecules include those molecules capableof binding known or yet to be discovered TAAs. Illustrative TAAs includep53 and Her-2 Neu.

As also discussed, the present polyspecific binding molecules alsoinclude an antibody binding domain such as a sc-Ab and particularly asc-Fv or functional fragment thereof. As also discussed, methods ofmaking and using various sc-Fv molecules have been described. See e.g.,the U.S. Pat. No. 5,637,481; Jost, C. R. et at supra, and Lindhofer, H.et at (1996), supra.

More particular sc-Ab molecules generally include immunoglobulin chainsthat are capable of specifically binding antigen. The immunoglobulinchains may include full-length immunoglobulin chains, e.g., afull-length V_(L) and V_(H) chain; or may include a functional fragmentof one or both full-length immunoglobulin chains. The term “functionalfragment” as used with respect to a sc-Ab means a portion of thefull-length immunoglobulin chain making up that sc-Ab that is capable ofspecifically binding at least about 70% and preferably at least about80%, 90%, or 95% up to about 100% of a specific antigen when compared tothe full-length immunoglobulin chain. Specific binding can bequantitated by a variety of techniques such as a Western blot or othersuitable antibody binding assay as described below.

More specific examples of an antibody binding domain in accord with theinvention include, but are not limited to, (1) a single variable regionof an antibody (V_(L) or V_(H)) 2) two or more single-chain variableregions (e.g. V_(L)+V_(H); V_(L)+V_(L); or V_(H)+V_(H)) or thecomplementary determining region (CDR) thereof. Each variable regionfragment (V_(L) or V_(H)) is preferably encoded by V_(L)+J_(L) or byV_(H)+D_(H)+J_(H) sequences and composed of approximately 100 aminoacids. Within these sequences are three regions of hypervariabilitycalled complementarity determining regions (CDR) that appear to containthe amino acids that line the antibody's combining site. The CDRs areinterspersed in four regions of lower variability called frameworkregions (FR).

In one embodiment, the antibody binding domain of a polyspecific bindingmolecule can be formed by the association of V_(L) and V_(H)polypeptides into a β-pleated sheet conformation, with the CDR regionscontained at, or near, the loops between strands. Occasionally, theV_(L)+V_(L) pairs or the V_(H)+V_(H) pairs or the V_(L) or V_(H) alonecan bind antigen.

In a more preferred embodiment, the antibody binding domain is a sc-Aband particularly a sc-Fv including at least one and preferably one ofthe following: (1) a V_(L) chain and a V_(H) chain; (2) a V_(L) chainand a V_(L) chain; (3) a V_(H) chain and a V_(H) chain; (4) a singleV_(L) chain; or (5) a single V_(H) chain. The binding domain may includeimmunoglobulin chains of any suitable isotype, e.g., IgG or IgM.

In embodiments in which the polyspecific binding region includes anantibody binding domain that exists as two variable regions linked as asingle chain protein such as a sc-Fv (e.g., V_(L)+V_(H); V_(L)+V_(L);V_(H)+V_(H),) the single chain protein will preferably include apolypeptide linker sequence to link the two variable domains together. Avariety of peptide linkers are known to be suitable for making sc-Fvconstructs. See e.g., Huston, J. S. (1988) PNAS (USA) 85:5879; andPluckthorn, A. (1992) Immunological Rev. 103:151. A specificallypreferred linker has the following general formula (Gly4Ser)_(n) inwhich n is from about 2 to 5 and preferably about 3.

A variety of immunoglobulin V_(H) and V_(L) chains have been describedat the nucleic acid and protein levels. See, e.g., Davis in FundamentalImmunology, (1993) supra; Kabat E. A., supra; U.S. Pat. No. 5,637,481;Jost, C. R. et al. supra, and Lindhofer, H. et al. (1996), and theBrookhaven Protein Data Bank (Brookhaven Protein Data Base, ChemistryDept. Brookhaven National Laboratory, Upton, N.Y. (1973).

As mentioned previously, a variety of sc-Fv constructs have beenreported. The constructs can be used in accord with the invention tomake a wide spectrum of polyspecific binding proteins. See generally,Pastan, I and Fitzgerald D., (1991) Science 254:1173; Webber, et al.,Molecular Immunol. (1995), 32:249; and published PCT application Nos.WO96/05228 and WO 97/28191 for disclosure relating to making and usingsingle-chain antibodies.

More specific sc-Fv molecules are those capable of specifically bindingcell surface targets such as glycoproteins and lipoproteins. Examples ofparticular glycoproteins include, but are not limited to, CD3/TcR andCD28. See Gilliland L. K., et al., (1996) Tissue Antigens 47:1 fordisclosure relating to generating and characterizing sc-Fv moleculesthat bind these molecules and other surface molecules. Additionalspecific sc-Fv constructs have been disclosed in Colcher, D., et al.(1990) J. Nat. Cancer Inst. 82:1191; and Yokota, T., et al. (1992)Cancer Res. 52:3402).

Additional sequence information relating to specific sc-TCR and sc-Abchains is available from the National Center for BiotechnologyInformation (NCBI)—Genetic Sequence Data Bank (Genbank) at the NationalLibrary of Medicine, 38A, 8N05, Rockville Pike, Bethesda, Md. 20894.Genbank is also available on the internet at http:www.ncbi.nlm.nih.gov,See Benson, D. A. et at (1997) Nucl. Acids. Res. 25: 1 for a descriptionof Genbank.

The Ig-C_(L) chain of a polyspecific binding protein of this inventionis κ- or λ-type immunoglobulin light chain region The κ-typeimmunoglobulin light chain constant region will sometimes be referencedherein as “Cκ chain”, whereas the λ-type immunoglobulin constant chainlight chain region will often be referred to as “C_(λ) chain”. Forexample, the Ig-C_(L) chain can be a Cκ chain or a suitable fragmentthereof such as those disclosed below. In addition, an Ig-C_(H) chain ofthe polyspecific binding protein can be μ, δ, γ, α, or ε type asdesired. Preferably the amino acid sequences of the immunoglobulin heavyand light chains are known.

As noted, the present polyspecific binding molecules are fullyfunctional and soluble. By the term “fully functional” or similar termis meant that the binding molecules can specifically bind othermolecules for which binding is intended. More specifically, a bindingmolecule of this invention is fully functional if the sc-TCR part of themolecule can specifically bind an pMHC or pHLA (or a portion thereof).The term also means that the sc-Fv part of the binding molecule canspecifically bind an antigen or portion thereof. Assays for detectingspecific binding between a polyspecific binding molecule of interest andthe pMHC (pHLA) or the antigen include Western blots and other standardassays such as those disclosed below.

By the term, “specific binding” or a similar term is meant a moleculedisclosed herein which binds another molecule, thereby forming aspecific binding pair. However, the molecule does not recognize or bindto other molecules as determined by, e.g., Western blotting ELISA, RIA,mobility shift assay, enzyme-immuno assay, competitive assays,saturation assays or other protein binding assays know in the art. Seegenerally, Ausubel, et al infra; Sambrook, et al, infra; Harlow andLane, supra and references cited therein for examples of methods fordetecting specific binding between molecules.

By the term “fully soluble” or similar term is meant that the fusionprotein is not readily sedimented under low G-force centrifugation froman aqueous buffer e.g., cell media. Further, a specific polyspecificbinding molecule of this invention is soluble if can remain in aqueoussolution at a temperature greater than about 5-37° C. and at or nearneutral pH in the presence of low or no concentration of an anionic ornon-ionic detergent. Under these conditions, a soluble protein willoften have a low sedimentation value e.g., less than about 10 to 50svedberg units. Aqueous solutions referenced herein typically have abuffering compound to establish pH, typically within a pH range of about5-9, and an ionic strength range between about 2 mM and 500 mM.Sometimes a protease inhibitor or mild non-ionic detergent is added anda carrier protein may be added if desired such as bovine serum albumin(BSA) to a few mg/ml. Exemplary aqueous buffers include standardphosphate buffered saline, tris-buffered saline, or other known buffersand cell media formulations.

Conventionally, there are several means for linking the polyspecificbinding molecules disclosed herein including cellular, genetic, chemicaland biochemical methods. As will be appreciated, certain polyspecificbinding molecules of this invention can be joined (crosslinked) bychemical cross-linking; natural cross-linking by disulfide bonds;natural association without disulfide bonds; and connecting by agenetically encoded peptide linker (Bird, R. E., et al. (1988) Science242; Huston et al., supra). For example, coupling between desiredpolyspecific binding molecules will include standard protein couplingreactions such as those generally described in Means, G. E. and Feeney,R. E. (1974) in Chemical Modification of Proteins, Holden-Day. See also,S. S. Wong (1991) in Chemistry of Protein Conjugation and Cross-Linking,CRC Press.

Additionally, it will be appreciated that the polyspecific bindingcomplexes of the invention can be modified in several well-known ways tosuit intended uses. For example, the complexes can bedisulfide-stabilized in accordance with known methods see e.g., thepublished PCT application no. WO/29350.

The present binding molecules can also be made by employing inertpolymers called dendrimers. In a more specific embodiment, a particulardendrimer, known as the Janice face dendrimer, can be used to join orcouple together portions of the present binding molecules. In onespecific embodiment of this invention, the sc-Fv molecule can be made toinclude a C-terminal cysteine residue that can be used to cross-link theantibody to the dendrimer particularly through disulfide bonds. ThescTCR would then be coupled to the dendrimer through free amine groups.A preferred resulting product is a stable polyfunctional dendrimermolecule. See Example 19 below.

As discussed above, the present invention features multi-chainpolyspecific binding protein comprising at least one sc-TCR and anantibody binding domain. In one embodiment, the binding protein isrepresented by the following general formula:

wherein,

a) A represents an antibody binding domain or functional fragment

thereof,

b) B1, B2 are each independently a joining molecule the same ordifferent,

c) C1, C2 are each independently —H or a protein tag; and

d) D represents at least one sc-TCR molecule or functional fragmentthereof.

With respect to the formula provided above, a single line represents acovalent bond (e.g., a peptide bond), whereas a double line representsone or more covalent bonds, e.g., a disulfide bond such as those linkingimmunoglobulin heavy chains; or the double line represents hydrogenbonds. The brackets indicate flexibility in the sequential arrangementof the bracketed molecules (i.e., subunits). Thus, the order of thesubunits is not important so long as each subunit performs the functionfor which it is intended.

In the formula shown above, the subunits A, B1, B2, C1, C2 and D eachindependently represent preferably one or a plurality of molecules. Ininstances where A or D represents a plurality of molecules, eachmolecule will preferably be attached to the same type of molecule (e.g.,sc-TCR fused to another sc-TCR, sc-Fv fused to another sc-Fv).Preferably each molecule in the plurality is spaced from another by asuitable peptide linker sequence. The number of linked molecules willvary depending on intended use but will generally be from between about2 to 10, preferably from about 2 to 5, more preferably 2 of suchmolecules, and most preferably 1 molecule. Each of the subunitsdescribed above can be fused directly to another subunit or it may bespaced therefrom by a suitable peptide linker, e.g., to enhanceflexibility or binding affinity.

In a particular embodiment of the multi-chain polyspecific bindingmolecule represented above, A represents an F(v) or sc-Fv molecule; Drepresents a sc-TCR molecule, and each of C1 and C2 is —H.

A variety of joining molecules can be used in accord with the presentinvention. For example, in one embodiment, each of B1, B2 in the aboveformula can represent an immunoglobulin chain or suitable fragmentthereof capable of forming a specific binding complex as determined,e.g., by RIA, Western blot or other suitable binding assay. In a moreparticular embodiment, each of B1 and B2 is derived whole or in partfrom an immunoglobulin heavy chain. In this embodiment, the joiningmolecules can be the same or different class (IgG, IgA, IgM, IgD, or IgEclass) provided that the molecules are capable of forming a specificbinding pair. In addition, joining molecules consisting of chimericimmunoglobulin heavy chains are within the scope of the presentinvention. Preferred joining molecules include full-lengthimmunoglobulin heavy chain (Ig-C_(H)) or fragments thereof such as C_(H)¹; C_(H) ¹-C_(H) ²; C_(H) ¹-C_(H) ³ and C_(H) ¹-C_(H) ²-C_(H) ³.Additionally preferred fragments are capable of forming at least onedisulfide bond with another suitable immunoglobulin chain or fragment.An especially preferred pair of joining molecules is a pair ofimmunoglobulin heavy chains having an IgG isotype.

In another embodiment, each of B1 and B2 is an immunoglobulin lightchain the same or different provided that the light chains are capableof forming a specific binding pair as determined by RIA, Westernimmunoblot or other suitable binding assay. Suitable immunoglobulinlight chain joining molecules may be full-length or fragments thereofand can be κ or λ type. As will be appreciated, a suitable fragment of ajoining molecule will be one that is capable of forming a specificbinding pair as determined by assays described herein.

Additionally, an immunoglobulin joining molecule in accord with theinvention may be of animal (e.g., a rodent such as a mouse or rat), orhuman origin or may be chimeric or humanized (see e.g., Morrison et al.,PNAS 81, 6851 (1984); Jones et al. Nature 321, 522 (1986)). Exemplaryjoining molecules include those capable of being specifically bound byanti-idiotype antibodies such as those disclosed below as well ascommercially available anti-idiotype antibodies. See e.g., Linscott'sDirectory (40 Glen Drive, Mill Valley Calif. 94941), and by the AmericanType Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Md.20852.

More specific examples of multi-chain polyspecific binding proteins arerepresented in FIG. 7A. In the figure, the binding protein includes asc-TCR linked to a first immunoglobulin heavy chain (Ig-C_(H)). Thefirst Ig-C_(H) is linked to a second Ig-C_(H) to form a specific bindingpair. The second Ig-C_(H) is the same isotype (IgG) as the firstimmunoglobulin heavy chain and is further linked to an F(v) produced bya hybridoma cell. The sc-TCR is further linked to an immunoglobulinlight chain produced the hybridoma cell. Additional multi-chainpolyspecific binding complexes (sometimes referred to as “chimericbispecific” molecules or antibodies) can be made by using otherhybridoma cells or other sc-TCR/Ig molecules.

As mentioned previously, the present invention also featurespolyspecific binding proteins that include non-immunoglobulin joiningmolecules. For example, each of B1 and B2 in the formula shown above canbe a polypeptide that includes (or consists of) a protein-proteinbinding motif such as, e.g., a helix-turn-helix or leucine zipper motif.Many examples of these binding motifs have been described and are knownin the field. See e.g., Horberg, et al., (1993) Science 262:1401;Kamtekar, et al., (1993) Science 262:1680; Harris, et al., J. Mol. Biol.(1996) 236:1356.

More specifically, each of B1 and B2 can be a polypeptide that consistsof a protein-protein binding motif that is capable of forming a specificbinding pair. For example, each of B1 and B2 can be a protein-proteinbinding motif of a transcription factor such as fos or jun. Morespecific examples of protein-protein binding motifs include birA(LXLIFEAQKIEWR; SEQ ID NO. 5), avidin (ARKCSLTGKWTNDLGSNMT; SEQ ID NO.6), EE (EEEEYMPME; SEQ ID NO. 8), 6×HIS (GMAHHHHHH; SEQ ID NO. 9),fos/jun, and (TPPPEPET; SEQ ID NO. 10). See Rhind, S. K. (1992) U.S.Pat. No. 5,354,554; Altman, J. D. (1996) Science, 274:94; Shatz, P.(1993) Biotechnology, 11:1138. See also Examples 1-3 below.

As discussed, the present invention provides polynucleotides that encodesingle- and multi-chain polyspecific binding proteins (or portionsthereof). In one embodiment, the polyspecific binding protein is encodedby a polynucleotide which can be RNA, DNA, or a chimera thereof.Typically, the polynucleotide will include or consist of a DNA sequence(segment) that encodes the binding protein.

For example, a polynucleotide according to the invention typicallyincludes an operably linked leader sequence to provide appropriate cellprocessing signals. The leader sequence can be fused to the 5′ end ofthe DNA sequence encoding the sc-TCR molecule. In particular, the leadercan be covalently linked to the 5′ end of the DNA sequence encoding theV-α chain, or in some embodiments, the V-β chain of the sc-TCR. In otherembodiments, the leader will be fused to the sc-Ab and particularly tothe sc-Fv. In a more specific embodiment, the leader sequence can befused to the V_(H) chain or the V_(L) chain of the sc-Fv. It will berecognized however that although a specific leader sequence is linked toa particular sc-TCR or sc-Fv sequence, the leader sequence can often beexchanged using recombinant techniques without a detrimental effect onthe processing of the fusion protein. Thus in one embodiment, the 5′ endof the V-α chain is covalently linked to the 3′ end of a suitable leadersequence.

A variety of specific leader sequences can be used with thepolynucleotides. In one embodiment, the leader sequence is from betweenabout 12 to 26 amino acid residues in length. In a specific embodiment,a DNA sequence designed for insertion into a bacterial expression vectorcan include a Pel B leader sequence. Alternatively, DNA segments forinsertion into mammalian expression vectors may include an Ig-C_(L)leader such as a mammalian Cκ leader sequence. An exemplary Cκ leader isprovided below.

Additionally provided are polynucleotides that encode at least a portionof a polyspecific binding protein and particularly certain multi-chainpolyspecific binding proteins represented in the formula shown above. Ina more particular embodiment, the portion is sufficient to encode atleast the A-B1 or D-B2 subunits. See Examples 8, 9 and 12 below.

Additional polynucleotides according to the invention include a DNAsequence that encodes a single-chain polyspecific binding protein ofthis invention. More specifically, the polynucleotide will usuallyinclude a promoter, translation initiation signal, and leader sequenceoperably linked to the sequence. For optimal expression in bacterialhosts, the promoter is preferably phoA and the leader is pelB from E.coli. If desired, the DNA sequence can further comprise a ribosomebinding site from a gene 10 sequence. For optimal expression ineukaryotic hosts, the promoter is preferably a cytomeglovirus (CMV)promoter operably linked to a CMV enhancer element and the leader is amouse kappa chain leader. By the term “operably linked” is meant agenetic sequence operationally (i.e., functionally) linked to apolynucleotide, or sequences upstream (5′) or downstream (3′) from agiven segment or sequence.

Further polynucleotides according to the invention encode at least onesc-TCR or at least one sc-Fv each independently fused to a DNA sequenceencoding a suitable bacteriophage coat protein. Preferably, thebacteriophage coat protein is a gene VIII or gene III protein. Methodsfor making and using the polynucleotides have been described in thepending U.S. application Ser. No. 08/813,781. See also U.S. Pat. No.5,759,817 for disclosure relating to construction and use ofbacteriophage fusion proteins.

More specific polynucleotides of this invention include a DNA sequencethat encodes an sc-TCR that includes a V-α chain covalently linked by asuitable peptide linker sequence to the N-terminus of a V-β chain.Preferably, the DNA sequence further encodes an antibody binding domainand particularly an sc-Fv as discussed above separated from the sc-TCRby a suitable peptide linker sequence.

Polynucleotides that encode the present polyspecific binding proteinscan be obtained from a variety of sources including polymerase chainreaction (PCR) amplification of publicly available DNA sequences. In oneembodiment, the polynucleotide is provided in a DNA vector capable ofexpressing the molecule in a suitable eukaryotic or prokaryotic cellexpression system. As discussed, polynucleotides of this invention mayinclude operably linked transcriptional elements such as a promoter,leader and optimal enhancer sequences to drive expression of the solublescTCR fusion protein in a desired cell expression system. Alternatively,the DNA vector may be selected to provide some or all of the controlelements.

The term “vector” as used herein means a nucleic acid sequence capableof being incorporated and replicated into a host cell typicallyresulting in the expression of a nucleic acid segment of interest e.g.,a polynucleotide encoding a polyspecific binding molecule as describedherein. The vectors can include e.g., linear nucleic acid segments orsequences, plasmids, cosmids, yeast artificial chromosomes (YACs),phagmids and extra chromosomal DNA. Specifically, the vector can berecombinant DNA. Also used herein the term “expression,” or “geneexpression”, is meant to refer to the production of the protein productof the nucleic acid sequence of interest including transcription of theDNA and translation of the RNA transcription. Typically, a DNA segmentencoding an sc-TCR fusion protein of the invention is inserted into thevector, preferably a DNA vector, to replicate the DNA segment in asuitable host cell.

More particular DNA vectors according to the invention will includecontrol elements that are selected to optimize expression in a host forwhich it is intended. For example, a DNA vector for use in a bacterialhost can include a promoter such as the trp operon promoter, lacpromoter, trp-lac promoter, lac^(uvs) or phoA promoter. Exemplarypromoters are those such as phoA which provide strong, regulatedexpression during slow induction conditions lasting about several hours(e.g., 2 to 10 hours). Under suitable culture conditions, most strongpromoters are capable of providing soluble fusion protein at levels upto and exceeding approximately 10% of the total host cell protein. Seethe pending U.S. application Ser. No. 08/813,781 for more disclosurerelating to preferred conditions for expressing fusion proteins thatinclude a sc-TCR in bacterial cells.

In some embodiments of the invention, a polynucleotide encoding apolyspecific binding protein of interest will be recombinantlyengineered into an appropriate DNA vector. For example, in embodimentswhere the desired TCR or immunoglobulin chain is PCR-amplified, theoligonucleotide primers are usually configured with suitable restrictionsites on both ends of the primers so that the polynucleotide can bereplaced with another desired DNA. Thus, a suitable DNA vector of theinvention is one in which the desired binding molecule can be readilyinserted in the vector. Sometimes, as when an sc-TCR/IgG or other fusionbetween the sc-TCR or immunoglobulin heavy chain or fragment thereof isdesired, the Ig-C_(L) chain or chain fragment will be encoded by thevector and will be fused to the DNA segment by ligation. In other cases,the Ig-C_(L) chain or the fragment will be fused to the DNA segmentprior to the ligation to the vector.

In general, preparation of the present polyspecific binding proteins canbe accomplished by specific procedures disclosed herein and byrecognized recombinant DNA techniques. For example, preparation ofplasmid DNA, DNA cleavage with restriction enzymes, ligation of DNA,introduction of DNA into a cell, culturing the cell, and isolation andpurification of the expressed protein are known techniques. Seegenerally Sambrook et al. in Molecular Cloning: A Laboratory Manual (2ded. 1989); and Ausubel et al. (1989), Current Protocols in MolecularBiology, John Wiley & Sons, New York.

More particular strategies can be employed to express the polyspecificbinding molecules described herein. For example, in one approach, apolynucleotide encoding a polyspecific binding protein of interest canbe incorporated into a DNA vector by known means such as by use ofenzymes to restrict the vector at pre-determined sites, followed byligation of the DNA into the vector. The vector containing the DNAsequence is then introduced into a suitable host for soluble expressionof the binding protein. Selection of suitable vectors can be empiricallybased on factors relating to the cloning protocol. For example, thevector should be compatible with, and have the proper replicon for thehost cell that is being employed. Further, the vector must be able toaccommodate the DNA sequence coding for the protein that is to beexpressed. Preferred vectors are those capable of expressing the solubleproteins in mammalian cells e.g., pcDNA3 available from InVitrogen. Seealso Sambrook et al., supra and Ausubel et al. supra for other suitablevectors for use in, mammalian, cells. Typically, DNA vectors designedfor expression in bacteria and encoding soluble fusion proteins will notinclude a full-length Cλ or Cκ intron although these sequences can beincluded in vectors designed for expression in mammalian cells capableof RNA splicing.

More preferred DNA vectors are designed to express the polyspecificbinding protein in eukaryotic cells, particularly mammalian cells. TheDNA vectors can be formatted for replication in a bacterial host ifdesired so that suitable amounts of the DNA vector can be obtained. Forexample, a DNA vector will usually include (i) an origin of replication(Ori) functional in E. coli; (ii) a selectable antibiotic resistancegene (e.g., Amp, Tet, Neo or Kan resistance); (iii) a strong viralpromoter such as the cytomeglovirus (CMV) promoter and optional CMVenhancer element, (iv) an Ig-C_(L) leader sequence, (v) a sc-TCRmolecule of interest, (vi) a full-length Ig-C_(L) intron linked to anIg-C_(L) exon, (vii) a growth hormone polyadenlyation sequence, e.g.,bovine growth hormone (bgh) poly A sequence and (viii) DNA encoding aselectable eukaryotic marker such as a strong viral promoter (e.g.,simian virus 40 (SV40) promoter) linked to the antibiotic resistancegene and fused to a viral polyadenlyation sequence (e.g., the SV40 polyAsequence). Alternatively, the DNA vector can include all of (i)-(v), and(vii)-(viii), above, without the full-length Ig-C_(L) intron linked tothe Ig-C_(L) exon of (vi). An exemplary Ig-C_(L) leader sequence is themouse kappa leader. An example of a full-length Ig-C_(L) intron and exonis the full-length Cκ gene.

An example of a specifically preferred DNA vector for expressing thepresent single-chain polyspecific binding proteins in mammalian cells isthe pSUN 27 vector illustrated in FIG. 5. Construction and use of thepSUN27 vector has been described previously in the pending U.S.application Ser. Nos. 08/813,781 and 08/943,086. The pSUN27 vector hasbeen deposited pursuant to the Budapest Treaty with the American TypeCulture Collection (ATCC) at 10801 University Boulevard, Manassas, Va.20110-2209. The DNA vector was deposited with the ATCC on Sep. 17, 1997and was assigned Accession No. 209276. The pSUN27 vector includes a CMVpromoter, murine light chain leader sequence, Kozak consensus sequence,and the murine Cκ gene intron and exon sequence. See also Near, et al.,Mol. Immunology. (1990) for more specific disclosure relating to themurine heavy chain sequence.

Additionally preferred are DNA vectors suited for joining a desiredsc-TCR or functional fragment to an immunoglobulin heavy chain orfragment. For example, in one embodiment, the DNA vector can bereplicated in a bacterial host if desired. In particular, the DNA vectorwill usually include (I) an origin of replication (Ori) functional in E.Coli; (ii) a selectable antibiotic resistance gene (Amp, Tet, Neo, orKan); (iii) a strong viral promoter such as CMV promoter and optionalCMV enhancer; (iv) a V_(H) chain or fragment; (v) a C_(H) chain; (vi) agrowth hormone polyadenlyation sequence, e.g., bgh poly A sequence; and(vii) strong viral promoter (e.g., SV40 promoter) linked to theantibiotic resistance gene and fused to a viral polyadenylation sequence(e.g., SV40 PolyA sequence).

An example of a specifically preferred DNA vector for joining a desiredsc-TCR to the immunoglobulin chains is pSUN7 shown in FIG. 8.

A DNA vector of the invention can be modified according to conventionaltechniques to optimize expression in mammalian cells. For example, theeukaryotic marker encoding the neomycin resistance gene of the pSUN27 orpSUN7 vector described above can be replaced by DNA encoding thethymidine kinase (TK) gene to facilitate expression of the sc-TCR fusionprotein in TK- (TK deficient) mammalian cells. The DNA vector can bemodified in other ways well-known in the art (e.g., changing promoters,antibiotic resistance genes, replacing the CMV promoter with a promoterobtained from an immunoglobulin, SV40, an adenovirus or papilloma viruspromoter to optimize sc-TCR fusion protein expression in a desiredmammalian cell. Alternatively, the DNA sequence encoding the sc-TCRprotein can be inserted into well-known vectors suitable for expressionin yeast or insect cells, as desired. See e.g. Ausubel, et al., supraand Summer and Smith, infra.

A DNA vector especially designed for replication and expression of adesired binding protein in bacteria includes e.g., (i) an origin ofreplication functional in E. coli and derived e.g., from pBR322,preferably from well-known pUC19 vectors; (ii) a selectable antibioticresistance gene, e.g., ampicillin and/or neomycin resistance gene; (iii)a transcriptional termination region, e.g., the termination region ofthe E. coli trp operon; (iv) a transcriptional promoter, e.g., a phoA,tac, tac-lac, lacZ, lacing, T7, or T3 promoter; (v) a leader sequence,e.g., a pelB or ompA leader; (vi) a DNA sequence encoding the sc-TCRfused to a desired sc-Fv through a suitable peptide linker sequence(vii) a transcriptional terminator, e.g., the T1T2 sequence from theribosomal RNA locus of E. coli. Alternatively, the vector can include(i)-(vii), above, except that the sc-TCR is provided with a fusedIg-C_(L) chain or fragment.

Suitable host cells can be transformed by a variety of methods includingretroviral transfer, viral or bacteriophage infection, calcium-,liposome-, or polybrene-mediated transfection, biolistic transfer, orother such techniques known in the art.

As noted previously, in some cases it may be desirable to express thepolyspecific binding protein in non-mammalian cells. For example,suitable host cells for expressing the fusion proteins in bacteriainclude cells capable of being readily transformed and exhibiting rapidgrowth in culture medium. Particularly preferred hosts cells include E.coli, Bacillus subtillus, etc. Other host cells include, yeasts, e.g.,S. cerevisiae and insect cells. Exemplary cells for insect cellexpression are those capable of being infected by a baculovirus such asSf9 cells. See also, Summer and Smith (1988) A Manual of Methods forBaclovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555, College Station,Texas.

Although in the examples which follow cells of mammalian origin areused, in principle, nearly any eukaryotic cell is useful in the practiceof the subject invention. Examples include primate cells, e.g., humancells such as fibroblast cells, and cells from other animals such asovine, porcine, murine, and bovine cells. Specific examples of mammaliancells include COS, HeLa, and CHO, cells.

In general, cell culturing conditions are employed in which stablytransformed or transfected cell lines are selected e.g., byincorporation of a suitable cell selection marker into the vector (e.g.,an antibiotic resistance gene or G418). Cells which express a desiredpolyspecific binding protein of this invention can be determined byknown procedures e.g., by ELISA assay using commercially availablemonoclonal antibodies which specifically bind the binding molecule at adesired site. Illustrative of such sites includes the V-α or V-β chainof the sc-TCR or the variable chain of an antibody binding site, e.g.,the V_(H) or V_(L) chain of the sc-Fv. Alternatively, in embodiments inwhich a polyspecific binding molecule includes an immunoglobulin heavychain, e.g., an sc-TCR/IgG molecule, a monoclonal antibody can be chosenwhich specifically binds the Cκ or Cλ chain (or fragment). Examples ofmonoclonal antibodies and suitable assays are provided in the examplesbelow.

If included, a leader sequence in a DNA vector suitably directsexpression of the binding protein to host cell membranes or to the hostcell media and can be formatted to include a restriction site so thatDNA encoding, e.g., a V-α chain of interest, can be conveniently ligatedto the construct. Suitably, the restriction site is incorporated intothe 3′-end of the leader sequence, sometimes referred herein as ajunction sequence, e.g. of about 2 to 10 codons in length; and linked tothe V-α chain so that the coding region for the V-α chain is typicallythe first amino acid of the V-α coding region. Alternatively, the leadersequence can be linked to the V_(H) or the V_(L) coding region of thesc-Fv chain. For example, one restriction site is the Sfi I site,although other cleavage sites can be incorporated before the V-α chaincoding region to augment convenient insertion of the V-α chain into thevector construct. As discussed above, use of such a restriction site incombination with a second restriction site, typically positioned at thebeginning of the V-α chain, enables rapid and straightforward insertionof sequences coding for a wide variety of V-α chains, or V-α, C-αchains. Preferred leader sequences contain a strong translationinitiation site and can sometimes include a cap site at the 3′-end oftheir mRNA. As mentioned above, exemplary leader sequences include pelB,and OmpA for bacterial expression and a Cκ mouse kappa chain leadersequence for mammalian expression.

The present invention also includes methods for isolating thepolynucleotides or vectors encoding same. In general, the methodsinclude introducing the vector or polynucleotide into desired hostcells, culturing the cells and purifying the encoded polyspecificbinding molecule (or portion thereof) from the host cells to obtainsubstantially pure protein. The vector or polynucleotide can also beisolated in substantially pure form by standard methods. Typically, thevector or polynucleotide will be DNA for most recombinant manipulations.

In some instances, the polyspecific binding proteins of the presentinvention will include one or more fused protein tags (typically one ortwo). For example, the protein tags can be used to help purify theprotein from naturally occurring cell components, which typicallyaccompany the fusion protein. In other cases, the protein tag can beused to introduce a pre-determined chemical or proteolytic cleavage siteinto the soluble fusion protein. Particularly, contemplated isintroduction of a segment encoding a protein tag into a DNA vector,e.g., between sequence encoding the soluble fusion protein and theIg-C_(L) chain or suitable fragment so that the scTCR molecule can becleaved (i.e. separated) from the Ig-C_(L) chain or fragment if desired.

Polyspecific binding molecules that include a protein tag can have thattag fused to the molecule by genetic or chemical manipulations asneeded. In one embodiment, the binding molecule includes one protein tagfused to the C-terminus of the protein. Alternatively, the protein tagcan be fused to the N-terminus of the binding protein. In anotherembodiment, the protein tag is fused between the sc-TCR and sc-Abmolecules of the polyspecific binding protein.

The polyspecific binding proteins of this invention can be purified byseveral conventional techniques. For example, as previously mentioned,the binding proteins can include at least one protein tag (the same ordifferent), including tags which comprise a chemical or proteasecleavage site. Particularly, a protein tag can be a polypeptide bearinga charge at physiological pH, such as e.g., 6×HIS. In this embodiment, asuitable synthetic matrix can be used to purify the fusion protein. Moreparticularly, the synthetic matrix can be a commercially availablesepharose matrix, such as e.g. Ni-Sepharose or other such suitablematrixes capable of binding the 6×HIS tag at about pH 6-9. Othersuitable tags include EE or MYC epitopes, which are specifically boundby commercially available monoclonal antibodies. In general, a widevariety of epitopes capable of being specifically bound by an antibody,e.g., a monoclonal antibody, are capable of serving as a protein tag.Other suitable synthetic matrices includes those with a bound antibodycapable of specifically binding the present sc-TCR proteins. Exemplaryprotein tags include those with an enterokinase, Factor Xa, snake venomor thrombin cleavage site. See e.g., published PCT application WO96/13593. See also Example 6-7 below.

An expressed polyspecific binding protein can be isolated and purifiedby known methods including immunoaffinity chromatography,immunoabsorption, immunoprecipitation and the like. Importantly, thepreparative procedures will not usually require prolonged isolationsteps to obtain significant yields of the fusion protein. In accordancewith the protein purification methods described more fully below, yieldsfor most polyspecific binding proteins are in the range of about 2 to 6milligrams per liter.

As discussed, the polyspecific binding proteins of this invention can beexpressed and purified by one or a combination of strategies. In oneapproach, a polyspecific binding protein such as a single-chain fusionprotein is expressed in a suitable cell. Preferably the binding proteinis expressed in the cell or cell media. A cell extract or host cellculture medium is obtained and then centrifuged. The resultingsupernatant can be purified by affinity or immunoaffinitychromatography, e.g. Protein-A or Protein-G affinity chromatography oran immunoaffinity protocol comprising use of an antibody thatspecifically binds the binding protein. Examples of such an antibody arecommercially available monoclonal antibodies capable of specificallybinding the sc-TCR, sc-Fv or other portion of the binding molecule suchas the protein tag or immunoglobulin heavy chain portion. More specificexamples of suitable monoclonal antibodies are those capable of bindinga V-α chain or v-β chain of the sc-TCR, e.g., H57, B20.1, MR5-2, andF23.1 (Pharmagen). Specific examples of such antibodies areanti-idotypic antibodies such as those in the examples below.

As described above, the polyspecific binding molecules of the presentinvention are provided in a soluble and fully functional form. Thus, inone embodiment, the binding molecules are stably secreted into culturemedium and are capable of specifically binding a ligand of interest suchas a TCR antigen or portion thereof capable of binding the bindingmolecule. In embodiments of the invention in which a polyspecificbinding molecules is present as a single-chain, the molecule ispreferably stable under physiological conditions in the substantial orcomplete absence of a chaotropic agent such as a detergent or the like.Thus, the binding molecules will usually not include regions rich inhydrophobic amino acids such as those amino acids found in a TCRtransmembrane region.

The polyspecific binding molecules provided herein can be modified bystandard methods to include a variety of covalently linked protein tags(effectors). For example, one or more effectors or tags can be added tothe binding molecules to visualize bridging between bound cells and/orto boost recognition, damage or killing by immune cells. Potential sitesfor adding the effectors or tags include the sc-TCR, sc-Fv orimmunoglobulin heavy chain portion (if present). Preferred tagsgenerally impart a desired biological, chemical or physical property.More specific effectors or tags have been described in the pending U.S.application Ser. Nos. 08/813,781 and 08/943,086.

Additional examples of suitable protein tags include polypeptidesequences that have a charge at physiological pH, such as, e.g., 6×HIS.In this instance, a suitable synthetic matrix to purify the polyspecificbinding complex would be, e.g., a commercially availablemetallo-sepharose such as, e.g., Ni-sepharose or other such suitablematrix capable of binding 6×HIS at about pH 6-9. The EE epitope and mycepitope are further examples of suitable protein tags, which epitopescan be specifically bound by one or more commercially availablemonoclonal antibodies. Effector molecules may be conjugated to thepolyspecific binding complexes by means of a heterobifunctional proteincross-linking agent such as, e.g., SPDP, carbodimide, or the like. SeeMeany and Feeney, supra; Wong, supra.

It will be useful for some applications to non-recombinantly modify thepolyspecific binding complexes of the invention by non-genetic means.For example, the binding complexes can include a variety ofpharmaceutical agents in addition to those described above such asdrugs, enzymes, hormones, chelating agents capable of binding, e.g., aradionuclide, as well as other proteins and polypeptides useful fordiagnosis or treatment of disease. For diagnostic purposes, thepolyspecific binding molecule can either be labeled or unlabelled. Forexample, a wide variety of labels may be suitably employed, such asradionuclides, fluors, enzymes, enzyme substrates, enzyme cofactors,enzyme inhibitors, ligands such as, e.g., haptens, and the like.

For some applications, it will be desirable to position a polyspecificbinding molecule by including a fused peptide linker sequence. Severalsuitable peptide linkers and methods of testing same have beendescribed. In some cases it may be useful to add an agent to the fusedpeptide linker in accordance with well-known techniques. Examples ofuseful agents include photometrically detectable labels such as, e.g., adye or a fluor; an enzyme (such as, e.g., β-galactosidase, alkalinephosphatase, or horseradish peroxidase; which enzymes are capable offorming a photometrically detectable label). See generally U.S. Pat. No.5,434,051 for a discussion of suitable photometrically detectablelabels. Alternatively, the agents can be conjugated directly to thepolyspecific binding molecules disclosed herein by a variety of othermeans not involving a peptide linker, some of which means are disclosed.

Further, the polyspecific binding proteins of the invention can bepost-translationally modified if desired by e.g., carbohydrate or fattyacid addition. For example, the binding molecules can be modified byglycosylation. Glycosylation sites on proteins are known in the art andare typically either N-linked (asparagine-linked) or O-linked (serine-or threonine-linked). Such glycosylation sites can be readily identifiedby inspection of the protein sequence. The present binding molecules canbe glycosylated by suitable eukaryotic cells as evidenced by, e.g.,SDS-PAGE gel electrophoresis. SDS-PAGE gel electrophoresis and otherrelated methods can be combined with conventional biochemical techniquessuch as, e.g., enzymatic digestion, to detect carbohydrate bound to thepolyspecific binding proteins of the invention. Examples of preferreddigestive enzymes include, e.g., endoglycosidases, and exoglycosidasesavailable, e.g., from New England Biolabs (Beverly Mass.). Accordingly,the polyspecific binding molecules of the invention can be readilyanalyzed for the presence of carbohydrate groups, particularlyoligosaccharide groups.

In some instances, it may be useful to obtain substantially purepolyspecific binding molecules of the invention in glycosylated form.Particularly, such glycosylated molecules may exhibit less in vivodegradation when administered as a therapeutic agent, thereby increasingcirculating half-life (see e.g., Goto, M. et al. Bio/Technology 6:67(1988)).

In particular, the present polyspecific binding molecules are alsosuitable for a variety of in vitro and in vivo uses including diagnosticand imaging applications as well as HLA typing. See e.g., A. K. Abbas,Cellular and Molecular Immunology, page 328 (W.B. Saunders Co. 1991).For in vivo imaging applications, a polyspecific binding protein ofinterest can be detectably labeled by addition of ¹²⁵I, ³²P, ⁹⁹Tc orother detectable tag. The labeled polyspecific binding molecule can thenbe administered to a mammal and the subject scanned by known procedures.Such an analysis of the mammal could aid in the diagnosis and treatmentof a number of disorders including e.g. undesired expression of APCsaccompanying immune system disorders.

Molecular weights of present polyspecific binding molecules will varydepending on a number of factors including whether a particular bindingmolecule includes one or more sc-TCR molecules, what specific antibodybinding domain is included, whether a full-length Cκ or Cλ chain ispresent, or whether one or more protein tags is employed. In general, inembodiments in which the polyspecific binding molecule is present as asingle-chain, the binding molecule will have a molecular weight frombetween about 80 to 110 kDA, and particularly from between about 90 to100 kDA. In this embodiment, the V-α and V-β chains of the sc-TCR willhave a molecular weight of greater than about 16 kDA, more typicallybetween about 12 to about 20 kDa. Additionally, in embodiments in whichthe sc-Fv includes a V_(H) and a V_(L) chain, the chains will have amolecular weight of greater than about 18, more typically about 12 toabout 20 kDA. The molecular weight of a specific binding molecule willdepend on several parameters including the number of sc-TCR or sc-Fvmolecules present.

As discussed, some polyspecific binding molecules of the presentinvention are multi-chain molecules and especially bispecific chimericantibodies. See FIGS. 7A and 7B. Typically, the multi-chain moleculeswill have a molecular weight from between about 150 to about 250 kDa orgreater depending, e.g., on the number of sc-TCR or sc-Fv moleculespresent. All of the above mentioned molecular weights are determined byconventional molecular sizing experiments such as SDS-PAGE gelelectrophoresis or centrifugation. See generally Sambrook, et al., supraHarlow and Lane, supra; Ausubel et al, supra.

In some settings it can be useful to increase the valency of aparticular polyspecific binding molecules. For example, one way toincrease the valency of a polyspecific binding molecule is to covalentlylink together between one and four binding molecules (the same ordifferent) by using e.g., standard biotin-streptavidin labelingtechniques, or by conjugation to suitable solid supports such as latexbeads. Chemically cross-linked proteins (for example cross-linked todendrimers) are also suitable polyvalent species. For example, theprotein can be modified by including sequences encoding amino acidresidues with chemically reactive side chains such as Cys or His. Suchamino acids with chemically reactive side chains may be positioned in avariety of positions in the fusion protein, preferably distal to thebinding region of the sc-TCR or sc-Fv.

As a specific example, the C-terminus of the polyspecific bindingmolecule can be covalently linked to a protein purification tag or otherfused protein which includes such a reactive amino acid(s). Suitableside chains can be included to chemically link two or more fusionproteins to a suitable dendrimer particle to give a multivalentmolecule. Dendrimers are synthetic chemical polymers that can have anyone of a number of different functional groups on their surface (D.Tomalia, Aldrichimica Acta, 26:91:101 (1993)). Exemplary dendrimers foruse in accordance with the present invention include e.g. E9 starburstpolyamine dendrimer and E9 comburst polyamine dendrimer, which can linkcysteine residues.

Highly useful in vitro and in vivo T-cell binding assays have beendisclosed in published PCT Application Nos. PCT/US95/09816,PCT/US96/04314 and PCT/US97/01617, as well as the pending U.S. patentapplication Ser. Nos. 08/382,454, 08/596, 387 and 08/943,086. Thedisclosed T-cell binding assays can be used or readily adapted ifnecessary to test the function of the polyspecific binding proteins ofthis invention. The disclosures of said published PCT application Nos.PCT/US95/09816, PCT/US96/04314, PCT/US97/01617, and pending U.S.application Ser. Nos. 08/382,454, 08/596, 387 are each incorporatedherein by reference.

The ability of a polyspecific binding protein of the present inventionto modulate activity of an immune cells and especially a T-cell (i.e.cause or elicit T-cell activity such as proliferation) can be readilydetermined in accordance with the assays and materials for performingthe assays disclosed in said published PCT Application Nos.PCT/US95/09816, PCT/US96/04314, PCT/US97/01617, as well as said pendingU.S. patent application Ser. Nos. 08/382,454, 08/596, 387 and08/943,086. See also Matsui, et al. (1994) PNAS (USA) (1994) 91:12862.

More specifically, as disclosed in said published PCT Application Nos.US95/09816, PCT/US96/04314, PCT/US97/01617, as well as said pending U.S.patent application Ser. Nos. 08/382,454, 08/596, 387 and 08/943,086, invitro assays can be performed to determine if a molecule is capable ofmodulating T-cell activity. Such assays can be modified to determinefunctionality of the polyspecific binding proteins. Generally, aexemplary assay is conducted as follows, by the sequential steps 1-4below. T-cells suitably express a marker that can be assayed and thatindicates T-cell activation, or modulation of T-cell activity afteractivation. Thus, as disclosed in the prior applications, the murineT-cell hybridoma D011.10 expressing interleukin-2 (IL-2) upon activationcan be employed. IL-2 concentrations can be measured to determine if aparticular sc-TCR fusion molecule is capable of modulating activity ofthe T-cell hybridoma (e.g., increasing IL-2 production). A generalexample of such a suitable assay is conducted by the followingsequential steps:

1. Suitable T-cell hybridomas or T-cells are obtained.

2. The T-cell hybridoma or T-cells are then cultured under conditionsthat allow proliferation.

3. The proliferating T-cell hybridoma or T-cells are then contacted withone or more of the polyspecific binding proteins. The cells willtypically not proliferate (i.e. they are resting) until the polyspecificbinding protein is added along with suitable target cells.

4. In cases where non-hybridoma T-cells are employed such as naïveT-cells, it may be useful to add a suitable co-stimulatory factor toprovide signals necessary for activation. The T-cell hybridomas orT-cells are subsequently assayed for a marker, e.g. IL-2 production ismeasured. In embodiments in which a bispecific molecule is used,production of IL-2 is one way to evaluate the extent to which thebispecific molecule can modify the T-cell response. Preferred arebispecific molecules that provide for cell bridging and facilitate abouta two to about a threefold increase in IL-2 over a suitable control(i.e. an unstimulated T-cell). Additionally preferred are bispecificmolecules which when used without added immune cells will not result instimulation and in significant IL-2 production as measured by theabove-mentioned general assay. That is, addition of the polyspecificbinding molecule without addition of target cells will not result insignificant T-cell stimulation (i.e. IL-2 production). See Example 14below for a more specific assay.

As disclosed previously in said published PCT Application Nos.PCT/US95/09816, PCT/US96/04314, PCT/US97/01617, and in said pending U.S.patent application Ser. No. 08/382,454, 08/596, 387 and 08/943,086, theT-cells employed in the assays are usually incubated under conditionssuitable for proliferation. For example, a DO11.10 T-cell hybridoma issuitably incubated at about 37° C. and 5% CO₂ in complete culture medium(RPMI 1640 supplemented with 10% FBS, penicillin/streptomycin,L-glutamine and 5×10-5 M 2-mercaptoethanol). Serial dilutions of afusion protein can be added to the T-cell culture medium inconcentrations typically in the range of from 10⁻⁸ to 10⁻⁵ M. T-cellactivation signals are preferably provided by antigen presenting cellsthat have been loaded with the appropriate antigen.

As disclosed previously in said published PCT Application Nos.PCT/US95/09816, PCT/US96/04314, PCT/US97/01617 and in said pending U.S.patent application Ser. No. 08/382,454, 08/596, 387 and 08/943,086,rather than measurement of an expressed protein such as IL-2, modulationof T-cell activation can be suitably determined by changes inantigen-dependent T-cell proliferation as measured by radiolabellingtechniques as are recognized in the art. For example, adetectably-labeled (e.g., tritiated) nucleotide may be introduced intoan assay culture medium. Incorporation of such a tagged nucleotide intoDNA serves as a measure of T-cell proliferation. This assay is notsuitable for T-cells that do not require antigen presentation forgrowth, e.g., T-cell hybridomas. It is suitable for measurement ofmodulation of T-cell activation for untransformed T-cells isolated frommammals. T-cell proliferation following contact with the fusion protein(only in the presence of peptide/MHC target cells) indicates that themolecule modulates activity of the T-cells and can suppress immuneresponse. The in vitro T-cell proliferation assay is preferred formeasuring the effects of fusion proteins on antigen-specific changes inT-cell colony expansion in vivo. Measurement of IL-2 production orT-cell proliferation can be employed to determine if the polyspecificbinding protein is capable of modifying T-cell activation.

Additionally preferred bispecific binding molecules include thosecapable of mediating CTL killing of desired target cells as determinedby a cytotoxicity assay such as a conventional chromium (Cr⁵¹) releaseassay. In a specific embodiment, the chromium release assay is used tomeasure CTL killing. The cell killing can be monitored and quantified ifdesired by a number of suitable means including measuring the releasedchromium. The chromium release assay is readily adaptable for use withnearly any polyspecific binding molecules disclosed herein and suitabletumor cell targets. Preferred are bispecific binding molecules that arecapable of releasing between at least about 10 to 15% lysis with respectto spontaneous release from suitable control cells. See Example 18 belowfor more specific information regarding the chromium release assay.

In general, suitable T-cells for the assays are provided by transformedT-cell lines such as T-cell hybridomas or T-cells isolated from amammal, e.g., a primate such as from a human or from a rodent such as amouse, rat or rabbit. Other suitable T-cells include: 1) T-cellhybridomas which are publicly available or can be prepared by knownmethods, 2) T helper cells, and 3) T cytotoxic cells, preferablycytotoxic CD8+ cells. T-cells can be isolated from a mammal by knownmethods. See, for example, R. Shimonkevitz et al., J. Exp. Med., (1983)158:303.

Related in vitro and in vivo assays for testing sc-TCR molecules havebeen described in said published PCT Application Nos. PCT/US95/09816,PCT/US96/04314, PCT/US97/01617 and in said pending U.S. patentapplication Ser. Nos. 08/382,454, 08/596,387 and 08/943,086. Such assayscan be readily adapted for use with the present polyspecific bindingmolecules as needed.

See Example 14 below for an especially preferred assay for detectingstimulation of T hybridoma cells using preferred bispecific hybridmolecules.

The present invention provides additional methods for testing thesingle- and multi-chain polyspecific binding proteins disclosed herein.For example, the functionality of the sc-TCR or antibody-binding portionof the binding molecules can be readily demonstrated by a variety ofspecific binding assays. Preferred binding assays monitor and preferablyquantitate specific binding between the antibody binding portion and adesired cell surface protein. Preferred specific binding assays includeWestern blotting, ELISA, RIA, mobility shift assay, enzyme-immuno assay,competitive assays, saturation assays, cytometric assays or otherprotein binding assays know in the art. Preferred are assays that arecapable of detecting a cell surface protein, e.g., a TCR, glycoproteinor other suitable molecule.

One preferred assay for analyzing the present polyspecific bindingmolecules is an ELISA assay. For example, in one embodiment, suitablehost cells expressing a desired bispecific hybrid molecule are screenedin an ELISA format using an antibody that is capable of specificallybinding the hybrid molecule. Preferred are bispecific hybrid moleculesthat include a protein tag such as an EE-tagged molecule. In thisinstance, the tagged molecules can be probed using commerciallyavailable antibodies that specifically bind the tag. The bound antibodycan be conveniently detected using standard ELISA, e.g., by binding asecond detectably-labeled antibody that binds the antibody recognizingthe EE-tag. Alternatively, the bispecific hybrid molecule may be probedwith an antibody that specifically binds the sc-TCR or the antibodybinding domain and particularly the sc-Fv.

The above-described ELISA assays can be used to detect and characterizenearly any of the polyspecific binding proteins disclosed herein.Additionally, the ELISA assays can be used to screen cells for capacityto express a desired polyspecific binding protein. See Example 3 andFIGS. 9A, 9B, 10A, 10B and 11 for results of illustrative ELISA assays.

Additionally preferred assays for analyzing the present polyspecificbinding proteins include Western immunoblots. Briefly, a particularpolyspecific binding protein such as a bispecific hybrid molecule can beseparated by conventional gel electrophoresis and transferred to asuitable support medium. The transferred blot can then be probed with awide variety of antibodies such as those that specifically bind thesc-TCR or antibody binding domain, e.g., the sc-Fv. Bound antibody canbe visualized by standard detection methods. See the Examples below andFIG. 12.

Additionally preferred assays for analyzing the present polyspecificbinding proteins involve flow cytometric analysis. For example, specificbinding between the sc-TCR or sc-Fv portion of a bispecific hybridprotein and a glycoprotein or other suitable marker expressed on a cellcan be determined by flow cytometric analysis. In a more particularexample, T hybridoma cells or other suitable cells that express the CD3molecule are contacted with the bispecific hybrid molecule underconditions conducive to forming a specific binding complex. The cellsare then washed and contacted with a detectably-labeled antibody (e.g.,biotinylated) specific for a V chain of the sc-TCR or a protein tagattached to the bispecific hybrid molecule (e.g., the EE tag). Astandard chromogenic assay is then performed using labeled streptavidinand spectrophotometric detection methods. Functionality of the sc-Fv canbe demonstrated by staining of the T-hybridoma cells. Non-specificstaining can be detected by a variety of methods including use of Thybridoma cells that do not express the CD3 molecule. For someapplications it may be useful to check the binding specificity byincluding a suitable antibody that can compete with the bispecifichybrid protein for binding to the cells. Preferred bispecific bindingproteins will exhibit from an increase in cytochrome from between about5 to 1000 fold and preferably between from about 10 to 100 fold whencompared to a suitable control. See Example 15 and FIGS. 17-19 forresults of a flow cytometric analysis.

As discussed, the present invention also features recombinantbacteriophages that include fusion proteins that comprise sc-TCR orsc-Fv molecules fused to a suitable bacteriophage protein or fragmentthereof. As discussed, sc-Fv fusion proteins comprising a bacteriophagecoat protein are known in the field. Methods for making and using sc-TCRfusion proteins comprising a bacteriophage coat protein have beendisclosed in pending U.S. application Ser. No. 08/813,781. See also U.S.Pat. No. 5,759,817. It will be apparent from the examples that followthat the disclosed methods can be adapted, as needed, to facilitatemanufacture of the present recombinant bacteriophages.

More particularly, the present recombinant bacteriophages display fusionproteins that each include a sc-TCR or sc-Fv linked to a bacteriophagecoat protein or fragment. Preferred recombinant bacteriophages of thisinvention are bispecific and feature the binding specificity of thesc-TCR and sc-Fv fusion proteins. As disclosed in the pending U.S.application Ser. No. 08/943,086, the scTCR fusion protein generallyincludes a bacteriophage coat protein or fragment thereof covalentlylinked to a V-α chain fused to a V-β chain preferably through a flexiblepeptide linker sequence. Preferably, the bacteriophage coat protein is abacteriophage gene III or gene VIII protein. The sc-Fv fusion proteintypically includes a bacteriophage coat protein or fragment covalentlylinked to the V_(H) or V_(L) chain preferably through a flexible peptidelinker protein.

As used herein “bacteriophage coat protein” includes the full-lengthcoat protein. A suitable fragment of that coat protein is capable offacilitating packaging of the scTCR or sc-Fv and displaying the scTCR orsc-Fv as a fusion protein component of the bacteriophage coat.Successful packaging can be demonstrated in several ways includingplaque assays that quantitate production of infectious particles. Morespecific disclosure relating to methods of making and using thebacteriophages can be found in Examples 21, 24, 26-28 below.

In one embodiment, the recombinant bacteriophages of this inventiondisplay scTCR and sc-Fv fusion proteins that each optionally include oneor more fused protein tags (typically one or two). Attachment of atleast one protein tag has several advantages including providing astraightforward way of purifying the bacteriophages from cell componentswhich can accompany it. Preferred are proteins tags that facilitatechemical or immunological recognition of the bacteriophage such as thosespecific tags described below. An especially preferred tag is the EEsequence.

In particular, the sc-TCR and sc-Fv fusion proteins of the recombinantbacteriophages of this invention can include nearly any sc-TCR or sc-Fvmolecule described herein. For example, the sc-TCR fusion protein caninclude covalently linked in sequence: 1) a V-α chain, 2) a suitablepeptide linker sequence, 3) a V-β chain 4) a C_(β)-chain and 5) and afirst bacteriophage coat protein or fragment. The sc-Fv fusion proteincan include covalently linked in sequence: 1) a V_(H) chain, 2) asuitable peptide linker sequence, 3) a V_(L) chain, and 4) a secondbacteriophage coat protein or fragment. The first and secondbacteriophage coat proteins can be the same or different depending,e.g., on the amount or quality of display desired.

In embodiments in which the recombinant bacteriophage includes fusionproteins that each comprise a sc-TCR or sc-Fv fusion protein, thatbacteriophage will sometimes be referred to herein as a “bispecificbacteriophage” or simply “bispecific phage”. Illustrative of suchbispecific phages include those that display a desired sc-TCR fused tothe bacteriophage gene VIII protein and the sc-Fv fused to the gene IIIprotein. However, in some cases, it may be useful to make recombinantbispecific bacteriophages that display the sc-TCR fused to the gene IIIprotein and the sc-Fv fused to the gene WIT protein.

As discussed, additional disclosure relating to the construction and useof the sc-TCR fusion proteins can be found in the pending U.S.application Ser. No. 08/813,781. In particular, the pending U.S.application Ser. No. 08/813,781 discloses an sc-TCR fusion protein thatincludes a C-β chain fragment covalently linked between the C-terminusof the V-β chain and the N-terminus of the bacteriophage gene IIIprotein. Optionally, a protein tag can be covalently linked to theC-terminus of the C-fragment and the N-terminus of the bacteriophagegene III protein. Also disclosed is an sc-TCR fusion protein thatincludes a first protein tag covalently linked between the C-terminus ofthe V-β chain and the N-terminus of the bacteriophage gene III protein,and a second protein tag covalently linked to the C-terminus of thefusion protein.

Additionally disclosed in the pending U.S. application Ser. No.08/813,781 is an sc-TCR fusion protein that includes covalently linkedin sequence: 1) a V-α chain, 2) a peptide linker sequence, 3) a V-βchain covalently linked to a C-β chain fragment, and 4) a bacteriophagegene VIII protein. Also taught is an sc-TCR fusion protein that includescovalently linked in sequence: 1) a V-α chain covalently linked to a C-αchain fragment, 2) a peptide linker sequence, 3) a V-β chain covalentlylinked to a C-β chain fragment, and 4) a bacteriophage gene VIIIprotein. In this embodiment, the sc-TCR may further include a firstprotein tag covalently linked to the C-terminus of the V-β chain and theN-terminus of the gene VIII protein, and a second protein tag covalentlylinked to the C-terminus of the fusion protein. Additionally, a proteintag may be covalently linked to the C-terminus of the C-β chain fragmentand the N-terminus of the gene VIII protein.

If desired, the present recombinant bacteriophages can be manipulated tohave valancies from between about 2 to 10 and preferably from betweenabout 2 to 3. That is, the bacteriophages can be formatted toinclude: 1) between from about 2 to 3 sc-TCR fusion proteins, 2) betweenfrom about 2 to 3 sc-Fv fusion proteins, or 3) between from about 2 to 3sc-TCR and sc-Fv fusion proteins. Such polyspecific bacteriophages arehighly useful, e.g., as when it is desirable to increase the avidity orbinding affinity of an sc-TCR or sc-Fv fusion protein displayed on thebacteriophage.

The present invention also provides methods for making the recombinantpolyspecific bacteriophages described herein. For example, in oneembodiment, bacterial host cells are transfected with polynucleotidesthat encode a sc-TCR or sc-Fv in which the encoded sc-TCR or sc-Fv isfused to a suitable bacteriophage coat protein or fragment. Alsocontemplated are polynucleotides that encode a functional fragment ofthe sc-TCR or sc-Fv. Also envisioned are polynucleotides that encodemultiple copies (i.e. about 2 to 5) of the sc-TCR or sc-Fv. Morespecific disclosure relating to making and using polynucleotidesencoding the sc-TCR fused to a suitable bacteriophage coat protein orfragment can be found in the pending U.S. application Ser. No.08/813,781.

The present recombinant bacteriophages can be produced by one or acombination of strategies. Preferred are methods that use bacterial hostcells such as E. coli that are conducive to the bacteriophagepropagation. In a particular embodiment, the host cells are transfectedwith a polynucleotide that encodes the sc-TCR fusion protein underconditions sufficient to display same as part of the bacteriophage coator capsid. The host cell can be infected at the same time or at latertime with a polynucleotide encoding the sc-Fv fusion protein underconditions that are also conducive to displaying the sc-FV fusionprotein on the capsid. Production of the polyspecific bacteriophages canbe detected and quantified if desired by a variety of conventionalmethods such as RIA, ELISA, Western immunoblot and affinitychromatography.

In another embodiment, the recombinant polyspecific bacteriophages ofthis invention are made by infecting suitable host cells with“monospecific” recombinant bacteriophages that independently carry thesc-TCR or sc-Fv fusion proteins described herein. More specificdisclosure relating to such bacteriophages can be found in the pendingU.S. application Ser. No. 08/813,781. See also U.S. Pat. No. 5,759,817.For example, in a more specific embodiment, the polyspecificbacteriophages can be made by first infecting suitable bacterial hostcells with a monospecific bacteriophage and then infecting the same hostcells with the other monospecific bacteriophage. Alternatively, theinfection can be conducted by co-infecting with both monospecificbacteriophages.

It will be appreciated that the present methods for making therecombinant polyspecific bacteriophages are highly flexible. That is,the order in which the host cells are transfected (or infected) with aparticular polynucleotide (or recombinant bacteriophage) is notimportant so long as the resulting recombinant bacteriophage has thebinding specifies intended.

The recombinant bacteriophages of this invention provide a number ofimportant uses and advantages. For example, the bacteriophagespreferably display full- or nearly full-length scTCR and sc-Fvmolecules. Accordingly, use of the present bacteriophage librariespositively impacts analysis of scTCR and sc-Fv molecules, particularlyscTCR and sc-Fv binding pockets.

The present recombinant bacteriophages are particularly useful for awide spectrum of screens such as those formatted to detect and evaluatespecific binding of a sc-TCR and sc-Fv molecules. The bacteriophages arealso useful for analyzing a variety of binding molecules such asantigens, antibodies, small molecules, superantigens and MHC/HLA peptidecomplexes. Importantly, the present bacteriophage display librariesexpress fusion proteins with a V-α and a V-β chain, thereby making thefusion proteins more fully representative of TCRs found in vivo.

Additionally, the present bacteriophages can be manipulated to maximizeformation of specific binding complexes between the bacteriophages anddesired binding molecules or even cells, thereby increasing detection ofthe binding molecules or cells which may be rare or weakly binding. Thebacteriophages of the invention are especially amenable to biopanningtechniques (e.g., cell panning and immunopanning).

As discussed, the recombinant bacteriophages and bacteriophage librariesof the present invention can be provided in kit form. The kit mayinclude recombinant bacteriophages displaying a single type of sc-TCRand sc-Fv. Alternatively, the kit may include a recombinantbacteriophage library in which case the library will preferably includea variety of different sc-TCR and sc-Fv fusion proteins. More specifickits further include pertinent host cells and/or reagents for detectingthe bacteriophages, e.g., antibodies and directions for using the kit.

The present invention also provides a variety of methods foradministering at least one polyspecific binding protein to a mammal andpreferably a rodent or a primate such as a human patient. For example,in one embodiment, there is provided a method for administering apolynucleotide that encodes a polyspecific binding molecule andespecially a single-chain polyspecific-binding molecule. Preferred arepolynucleotides that can express the single-chain binding molecule inthe mammal. Preferably, DNA carrying the coding regions of the bindingprotein, suitably under the control of an strong eukaryotic promotersuch as a strong viral promoter (e.g., CMV), is injected directly intoskeletal muscle of the subject according to known methods. Methods foradministration of plasmid DNA, uptake of that DNA by cells of theadministered subject and expression of protein has been reported (see J.Ulmer et al. Science, (1993) 259:1745-1749). In embodiments in which thepolyspecific binding molecule is administered to a mammal and especiallya human, it is preferred that the isotype of the molecule be compatiblewith the host employed.

As noted previously, the polyspecific binding proteins of the presentinvention have therapeutic applications. For example, as discussed, thebinding molecules can be used to redirect the specificity of a certainimmune cells and particularly a T-cell, CTL, CD8+ cell, NK cell, ormacrophage to eliminate a desired target cell that expresses an MHC suchas a virally infected or tumor cell. Cross-linking of the immune cellswith the target cells provides a potent immune response sufficient todamage or kill the target cell.

Additionally, the polyspecific binding proteins described herein can beadministered to reduce or eliminate an immune response in a mammal,e.g., to treat a mammal such as a human that suffers from or issusceptible to cancer and an infectious disease. Also suitable fortreatment are those subjects suffering or likely to suffer from anundesired immune response e.g. patients undergoing transplant surgerysuch as transplant of heart, kidney, skin or other organs. In situationsinvolving transplant rejection, a treatment protocol may suitably becommenced in advance of the surgical procedure.

Administration of the polyspecific binding molecules described hereincan be via any suitable means such as administration of atherapeutically effective amount of the fusion protein or polynucleotideencoding same. In some embodiments in which DNA administration isdesired it may be helpful to provide two or more polynucleotidesencoding parts of a desired polyspecific binding protein such as whenuse of a bispecific hybrid molecule is desired.

A number of specific approaches can be employed to reduce or killdesired target cells in accord with the present invention. For example,one treatment method for damaging and preferably killing target cellsprovides for the administration of a therapeutically effective amount ofa desired polyspecific binding molecule to link target cells expressingan MHC complex to specific immune cells expressing a cell surfaceantigen. Association between the target cells and the immune cellsfacilitates an immune reaction that damages and preferably eliminatesthe target cells. In some embodiments, more than onepolyspecific-binding molecule may be administered as needed. In someinstances, T-cell mediated immune responses such as T-cellproliferation, differentiation, activation or B lymphocyte stimulationcan be selectively controlled.

The polyspecific binding proteins described herein can be administeredto a mammal by injection, e.g., intraperitoneal or intravenousinjection. In preferred embodiments, the polyspecific binding proteinsare preferably produced from mammalian or other suitable cells andpurified prior to use so it is essentially or completely free ofpyrogens. The optimal dose for a given therapeutic application can bedetermined by conventional means and will generally vary depending on anumber of factors including the route of administration, the patient'sweight, general health, sex, and other such factors recognized by theart-skilled.

Administration can be in a single dose, or a series of doses separatedby intervals of days or weeks. The term “single dose” as used herein canbe a solitary dose, and can also be a sustained release dose. Thesubject can be a mammal (e.g., a human or livestock such as cattle andpets such as dogs and cats) and include treatment as a pharmaceuticalcomposition which comprises at least one polyspecific binding proteinand typically one of such protein. Such pharmaceutical compositions ofthe invention are prepared and used in accordance with procedures knownin the art. For example, formulations containing a therapeuticallyeffective amount of the binding protein may be presented in unit-dose ormulti-dose containers, e.g., sealed ampules and vials, and may be storedin a freeze dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, e.g. water injections, immediately prioruse. Liposome formulations also may be preferred for many applications.Other compositions for parenteral administration also will be suitableand include aqueous and non-aqueous sterile injection solutions whichmay contain anti-oxidants, buffers, bacteriostat and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents.

Methods of the invention which include reducing or eliminating targetcells expressing e.g., a tumor or viral peptide loaded MHC. The methodsmay be used in combination with other therapies such as anti-viral,immunosuppressive, anti-cancer or anti-inflammatory therapies to providea more effective treatment regimen. For example, the polyspecificbinding proteins of this invention can be used with specific anti-viralagents such as those used to reduce or eliminate a retrovirus infectionand particularly infection by the AIDS virus. Additionally, thepolyspecific binding proteins can be used with standard anti-cancertherapies such as chemotherapy or immunotherapy.

As mentioned previously, in some instances it may be useful to produceantibodies to the polyspecific binding proteins described herein orfragments thereof. More specific methods for making antibodies have beendescribed in the pending U.S. application Ser. Nos. 08/813,781 and08/943,086.

As mentioned above, the polyspecific binding molecules described hereincan be readily modified by one or a combination of strategies to improvebinding. More specific disclosure relating to methods for improving thebinding of sc-TCR molecules has been reported in the pending U.S.application Ser. No. 08/813,781.

Substantially pure soluble fusion proteins or nucleic acids are at leastabout 90 to 95% pure and preferably at least 98% to 99% or more pure forpharmaceutical use. Once purified partially or to substantial purity,the soluble fusion proteins can be used therapeutically (includingextracorporeally), or in developing or performing in vitro or in vivoassays as disclosed herein.

All documents mentioned herein are fully incorporated herein byreference in their entirety. The following non-limiting examples areillustrative of the invention.

Example 1 Construction of p-149 Single-Chain (sc) TCR

The T cell clone, p-149, recognizes a peptide fragment (STPPPGTRV, SEQID NO. 11) of the human wild-type tumor suppresser protein p53restricted by HLA-A2.1. (See Theobald et al., PNAS, 1995) The T cellreceptor gene was cloned into a three domain single-chain formatpreviously shown to produce soluble TCR and functional receptormolecules (FIG. 1A).

In brief, mRNA was isolated from the T cell clone and cDNA was madeusing the Marathon cDNA Amplification Kit (Clontech). The cDNA was usedas a template in polymerase chain reaction (PCR) with primers KC171 andKC174 to produce a 5′SfiI3′SpeI Vα chain fragment including the firstseven amino acids of the Cα chain N-terminus. The same cDNA was thenused as a PCR template with primers KC172 and KC176 to generate a5′XhoI-3′XmaI V beta C beta chain fragment. The C beta chain wastruncated just before the cysteine residue at amino acid 127 of thefull-length C beta chain.

The alpha and beta chain fragments were cloned into the pGEM-T EasyVector System (Promega) for DNA sequence determination. Correctfragments were restriction digested and cloned into the expressionvector pKC60 to create a V alpha-(G₄S)₄ V beta C beta scTCR molecule.The pKC60 vector is referred to herein as PSUN23 (FIG. 3). The pKC60vector has been described in the pending U.S. application Ser. No.08/813,731. The new vector was named pNAG2 (FIG. 4).

The E. coli DNA construct pNAG2 was then reamplified by PCR with primersKC203 and KC208 to generate a 5′AgeI-3′HpaI/BspEI/NruI/ClaI DNAfragment. The scTCR fragment was cloned into the pGEM-T Easy VectorSystem for DNA sequence determination.

This new pGEM-based vector was then used as a “shuttle vector” forintroduction of other DNA fragments to create a bispecific sc molecule.

1. Cloning and expression of variant p-149 scTCR forms in E. coli.

It is possible to provide the p-149 sc-TCR in a variety of usefulconstructs. For example, four variations of the pSUN21 constructdescribed below can be used to express the scTCR. It has been found thatthe level of soluble scTCR is increased when the scTCR is expressed inthe pSUN21 scTCR design shown in FIG. 1B. Therefore, an initial cloningwill be accomplished by using this single-chain construct as a template.As described, a two-step cloning procedure will be used to assemble thescTCR into the expression vector. As discussed above, the p-149 cDNAencoding the full length alpha and beta chains of this receptor has beencloned. Related cloning methods can be used to make the variants.

For example, one variant of the p-149 TCR construct will closelyresemble the DO11.10 scTCR cloned into vector pSUN21. This constructcontains the Vα domain, a stretch of 10-25 amino acids followed by a(G4S)₄ linker, and the Vβ/Cβ domains. An EE-tag sequence will beincluded at the carboxyl terminal region. This facilitates detection ofthe molecule on immunoblots and can be used for cross-linking scTCRmolecules. A slightly modified second construct will encode a BirA site(see Example 24 below) at the carboxyl terminal end. BirA has beencharacterized as a biotinylation sequence and has been used to producetetrameric forms of MHC molecules. See Altman et al., Science, 274,94-96 (1996). The site will be used for constructing tetrameric scTCRmolecules for evaluation of the scTCR in cell binding and blockingassays. Also envisioned is construction of monomeric forms bycross-linking the scTCR with the scFv containing the BirA and avidin(see Example 24 below) tags, respectively. The addition of the BirA sitethrough genetic manipulation has an advantage over more traditionalbiotinylation methods that rely on chemical cross-linking protocols. Inmany instances, the use of such coupling agents results in thedenaturation of the protein which could be avoided by encoding at thegene level a site for biotinylation. Another advantage of having theBirA site is that stoichiometrically, a one:one molar ratio ofscTCR:scFv can be assembled.

In another example, a p-149 sc-TCR variant can be made that will containthe DNA encoding for the jun sequence. This will be cloned as a 3′DNAfragment into the scTCR design. The scTCR/jun fusion will be availablefor cross-linking with the scFv/fos fusion.

In yet another example of a p-149 variant, a fusion protein can be madewhereby, the carboxyl terminal region of the Cβ/EE-tag is geneticallyfused to pVIII, the major coat protein of filimentous phage. A varietyof sc-TCR fusions comprising bacteriophage proteins including the pVIIIand pIII proteins have been disclosed in the pending U.S. applicationSer. No. 08/813,781. As we described, the construction of bispecificphage (expression of both scTCR and scFv fragments on the surface of thephage) will be the one form of this hybrid molecule. The molecule has avariety of important uses including killing tumor cells in vitro and invivo, by forming a “bridge” between CTL and target cells. The pSUN21vector will be used to clone the scTCR/pVIII fusion. The vector has alacZ promoter and has been used in the development of the scTCR/phagedisplay model discussed in the preliminary results section. This is amodified pBluScript vector that can produce phage expressing scTCR/pVIIImolecules after superinfection with wild-type phage.

As disclosed in the pending U.S. application Ser. Nos. 08/813,781 and08/813,781, a variety of specific DNA vectors can be used to fuse adesired sc-TCR to bacteriophage coat proteins. For example, the pendingU.S. Applications disclose the DNA vectors pKC46 (pSUN18) and pKC62(pSUN19). These vectors have been deposited pursuant to the BudapestTreaty with the American Type Culture Collection (ATCC). The DNA vectorswere deposited with the ATCC on Feb. 26, 1997 and were assignedAccession Nos. 97895 (pSUN18) and 97896 (pSUN19). The DNA vector pKC62(pSUN19) includes a phoA promoter, modified pelB sequence, gene 10ribosome binding site and bacteriophage gene VIII protein. The DNAvector pKC46 (pSUN18) includes the lac Z promoter, an EE tag andbacteriophage gene III protein. The DNA vectors can be propagated in E.coli or other suitable host cells in accordance with standard methods.

The DNA vectors pKC46 (pSUN18) and pKC62 (pSUN19) are designed toaccommodate a variety of Vα, Vβ-Cβ and polypeptide linker sequences. TheVα chain of both DNA vectors can be removed by restriction digestionwith SFiI and SpeI. The Vβ-Cβ chain can be removed by restrictiondigestion with XhoI-XmaI. Additionally, the DNA vectors allow exchangeof the peptide linker sequence by restriction digestion with SpeI andXhoI. See FIGS. 2A-2E for more specific examples of sc-TCR constructs.

Example 2 Purification and Characterization of the p-149 sc-TCR

The pending U.S. application Ser. No. 08/943,086 discloses a variety ofmethods for purifying sc-TCR proteins including these that comprise theD011.10 sc-TCR. These methods can be adapted to purify the p-149 fusionprotein. For example, to purify the scTCR, an antibody with specificityfor a conformational epitope on Vβ 11.0 or Vα 2.3 can be used alonglines disclosed in the pending U.S. Application. In particular, thep-149 scTCR can be purified on an immunoaffinity column using thefollowing procedure.

Cell paste generated from a fermentor can be suspended in extractionbuffer followed by mechanical lysing of cells by passage through aFrench press. The supernatant is clarified by centrifugation at 25,000×gand applied to a Q-sepharose column. The scTCR is collected in theflow-thru and then applied to a Protein-A-sepharose column cross-linkedwith mAb H57-95. This is a hamster mAb specific for an epitope on theC-beta domain of murine TCRs. This antibody shows good bindingcharacteristics for murine TCRs and has been previously used to purifyintact scTCR molecules as well as breakdown products from the lysate. Toremove the degraded or improperly folded receptors, a second antibodyaffinity column will be used that can discriminate between scTCR that isconformationally intact from scTCR that has been degraded. Bound scTCRis eluted using a 50 mM glycine buffer, pH 11, and the scTCR preparationwill be analyzed by running sample on a 12% SDS polyacrylamide gel andstaining with coomassie brilliant blue or western blotting.

To determine whether the expressed protein is functional, the scTCR canbe tested in accord with assays disclosed in the pending U.S.application Ser. Nos. 08/813,781 and 08/943,086 such as a cell bindingassay and a blocking assay. The cell binding assay can be performed asdiscussed in the pending U.S. applications. Alternatively, the assayscan be modified by forming tetramers using scTCR molecules that includea single biotin sequence at the carboxyl terminal end. See Example 24below. The tetramers will be formed by adding streptavidin coupled to PEand then incubating these molecules with tumor cells known to naturallyprocess and present the 149 peptide associated with HLA-A2.1. Controlswill include cells only expressing HLA-A2.1. antigen and cellsexpressing neither the HLA-A2.1. nor the peptide. It is anticipated thata peak shift in fluorescence of cells expressing the peptide associatedwith HLA-A2.1.

Example 3 Construction, Expression and Characterization of the DO11.10scTCR

The DO11.10 TCR recognizes OVA peptide (323-339) in the context of theclass II MHC IA^(d) molecule. (See Haskins et al., J. Exp. Med., 1983.)The E. coli DNA construct pKC60 was reamplified by PCR with primersKC169 and KC208 to generate a 5′AgeI-3′HpaI/BspEI/NruI/ClaI DNAfragment. The scTCR DNA fragment was cloned into the pGEM-T Easy VectorSystem for DNA sequence determination. The correct scTCR DNA was thenrestriction digested with AgeI and HpaI and cloned into the “shuttlevector”, replacing the previous scTCR DNA fragment, to generate a newscTCR/scSc-Fv bispecific sc molecule. The DO11.10 bispecific sc moleculewas then cloned into pSUN27 to create pBISP/DO11.10 (FIG. 6).

The pBISP/DO11. 10 vector (pSUN 28) has been deposited pursuant to theBudapest treaty with the ATCC on Sep. 3, 1998 and was assigned AccessionNo. 203186.

1. Expression of variant scTCR molecules in E. coli.

The effect of changes in the design of the scTCR was investigated on thelevel of protein expression. Vectors which encode for the differentscTCR and fusion constructs were used to transform E. coli K91 cells.Expression experiments were carried out by growing transformed K91 cellsovernight in media containing inorganic phosphate to prevent activatingthe phoA promoter and inducing protein expression. The following morninga new culture was started from the overnight culture and grown untilphosphate had been depleted. The duration of induction was normalized bymonitoring the depletion of phosphate over time in the culture. See thepending U.S. application Ser. Nos. 08/813,781 and 08/943,086 foradditional disclosure relating to producing sc-TCR fusion molecules.

To compare the level of expression between the different constructs,protein was prepared from samples for analysis from cell lysates thathad been normalized to the same absorbence reading at 600 nm (10 OD/ml).Protein was released from cells by sonication and the sample wasclarified by centrifugation at 25,000×g for 20 minutes. Samples werethen loaded onto a 12% SDS-PAGE gel and after electrophoresis andtransfer of proteins to a nylon membrane, the TCR was detected byprobing with an antibody specific for the EE-tag. We observed in thisexpression experiment that alterations to the basic design of the scTCRcan produce significant changes in the level of soluble proteinexpressed. For example, the scTCR construct pSUN22, which includes theVα and Vβ domains joined by a synthetic linker, is not detectable in thesoluble fraction at the concentration of material loaded. A signal canbe detected by loading 50-fold more sample although the signal is stillnot equivalent to the levels seen with pSUN21. These data indicate highlevels of soluble scTCR can be produced in E. coli by modifying theconstruct design.

2. Characterization of the Soluble sc-TCR

A. Immunoprecipitation

The folding integrity of the scTCR protein produced by pSUN23 and pSUN19 DNA vectors was analyzed by running binding assay experiments usingtwo mAb (MR5-2 and F23.1) with specificity for correctly folded epitopeson Vβ 8.2. Furthermore, scTCR having correctly paired Vα with Vβ chainswere assayed using an anti-idiotype mAb, KJl, generated against theDO11.10 TCR. The binding assay experiments have been described in thepending U.S. application Ser. Nos. 08/813,781 and 08/943,086. The dataindicate that the scTCR protein has a conformationally correct Vβ domainand correctly paired Vα and Vβ domains.

B. Enzyme-Linked Immunoassay (ELISA)

A sandwich ELISA assay was used to further characterize the foldingdomains of the scTCR. Use of the ELISA assay is more fully described inthe pending U.S. application Ser. Nos. 08/813,781 and 08/943,086.Briefly, different dilutions of the scTCR was captured by anti-EE tagmAb coated on wells and was detected using one of the following mAbs,H57 (Cβ) MR5-2 (Vβ 8.2), F23.1 (Vβ 8.2) and KJl (Vα/Vβ). These datasupport the presence of a correctly folded scTCR and indicated that thescTCR is stable even after elution at high pH (11.0) and storage at 4°C. for several weeks.

C. Surface Plasmon Resonance (BioCore) binding studies using antibodiesand superantigen.

The scTCR/geneVIII fusion protein was characterized using surfaceplasmon resonance. The technique is more fully described in the pendingU.S. application Ser. No. 08/813,781. As disclosed in the pending U.S.application Ser. No. 08/813,781, the data indicate that although the twoanti-TCR mAbs recognize different epitopes they showed stearic hindrancewhich prevented binding of both mAbs to the beta chain in this assayformat. To demonstrate the presence of the bacteriophage pVIII proteinon the scTCR fusion protein, the scTCR was bound by the anti-M13antibody. Binding of the Streptococcus SAg known as SEC3 (ToxinTechnology, Tampa Fla.) to the scTCR/gene VIII fusion is also disclosedin the pending U.S. application Ser. No. 08/813,781. These data togetherwith the antibody binding data demonstrate that the E. coli producedscTCR is correctly folded.

3. Purification of the D011.10 scTCR

Methods for purifying sc-TCR fusion proteins have been disclosed in thepending U.S. application Ser. Nos. 08/813,781 and 08/943,086. TheD011.10 sc-TCR can be purified by those methods including the followingspecific method.

The fusion protein encoded by vector pSUN23 was purified fromtransformed cells by immunoaffinity chromatography in accordance withconventional methods. Briefly, the purification was performed by makingan affinity column by coupling 4 mg of anti-idiotype antibody, KJl perml of protein-A coated sepharose beads (Pharmacia). E. coli lysates wereprepared by solubilizing 50 g of fermentor-derived cell paste in 600 mlof solubilization buffer. Resuspended cells were lysed by two passagesthrough a French press. Insoluble material was removed by centrifugationat 27,000 g for 30 minutes and the supernatant was applied to aQ-sepharose anion exchange column. The scTCR protein was collected inthe flowthru and subsequently applied to the antibody column. BoundscTCR was eluted with a 50 mM glycine buffer, pH 11.0 and fractionscontaining protein were used for characterization.

The scTCR protein preparations were evaluated for purity byelectropheresis on an SDS-PAGE gel followed by commassie brilliant bluestaining. Protein integrity was determined by immunoblotting usingeither antibody H57-597 or anti-Glu-Glu (EE) tag antibody as a probe.Finally an aliquot of purified scTCR was run under reduced andnon-reduced conditions and after transfer of proteins the membrane wasprobed with the anti-EE tag antibody. The western blot results indicatedthat the purified scTCR was present as a monomer since both reduced andnon-reduced samples migrated with the 46 kD molecular weight marker.

Example 4 Construction of 145-2C11 sc-Fv

The anti-murine CD3-epsilon monoclonal antibody hybridoma cell line145-2C11 was purchased from American Type Culture Collection. (See Leoet al., PNAS, 1987) The DNA sequence of the variable chain codingregions of the antibody are available via the world wide web.

The Sc-Fv was designed as a V_(L)-linker-V_(H) gene construct. (See Jostet al, J. Biol. Chem., 1994) First, a shorter (G₄S)₃ linker was designedand made by annealing complementary oligos KC245 and KC246 to form a5′SpeI-3′XhoI DNA fragment. pKC60 was restriction digested with theappropriate restriction enzymes to drop out the previous linker DNAfragment and allow for ligation with the annealed oligos.

To prepare DNA encoding the V regions of the mAb, mRNA from 10⁶ 145-2C11 hybridoma cells was isolated using the RNeasy Total RNA Kit (Qiagen)in accordance with the manufacturer's instructions. V_(H) chain cDNA wasmade by incubating a mixture containing “back” primer KC244 along withthe 145-2C 11 mRNA. Standard amounts of nucleotides and reversetranscriptase were added to the mixture to form cDNA. The VH chain cDNAwas made in a similar manner with the exception that “back” primer KC253was used instead of the KC244 primer. VL chain cDNA was used as atemplate with primers KC243 and KC244 in a PCR reaction to amplify a 320bp 5′SfiI-3″SpeI V_(L) chain fragment. V_(H) chain cDNA was used as atemplate with primers KC247 and KC253 in a similar manner to amplify a350 bp 5′XhoI-3″XmaI VH chain fragment.

The V_(L) and V_(H) chain fragments were cloned into the pGEM-T EasyVector System for DNA sequence determination. Correct fragments wererestriction digested and cloned into the pKC60 expression vector alreadycontaining the shorter linker sequence described above.

Once the 145-2C11 Sc-Fv was complete, the DNA construct was reamplifiedby PCR with primers KC250 and KC251 to generate a 5′BspEI-3′NruI DNAfragment. The fragment was cloned into the pGEM-T Easy Vector System forDNA sequence determination. The correct DNA was then restrictiondigested and cloned into the “shuttle vector” downstream of the scTCR.See FIGS. 1C-1D for illustrations of the 145-2C11 sc-Fv (IC) and F23.1sc-Fv (ID) molecules.

Example 5 Design of the sc Molecule Linker Sequence

To connect the scTCR and scSc-Fv together as a single-chain fusionprotein, two different linker sequences were designed. One set ofannealed oligos, KC209 and KC210, coded for part of the CH1 domain ofmurine heavy chain followed by the standard (G4S) sequence. A second,shorter linker sequence was designed similarly but without the CH1domain using annealed oligos KC295 and KC296. Oligos were annealed togenerate a 5′HpaI-3′BspEI DNA fragment. The “shuttle vector” wasdigested with the appropriate restriction enzymes to drop out theprevious linker DNA fragment and allow for ligation of either of the twonew linker sequences between the scTCR and the Sc-Fv.

Example 6 Addition of a 3′ Peptide Tag to sc Molecule Construct

In the “shuttle vector” design outlined above, a stop codon and splicesite were introduced between the NruI and ClaI restriction sites as partof the PCR amplification of the scTCR with “back” primer KC208. To aidin downstream purification of the bispecific sc protein, a set ofannealed oligos (KC237 and KC238) was designed to introduce a 3′ EE tag(EEEEYMPME; SEQ ID NO. 8) with stop codon and splice site. The annealedoligo pair was cloned 5′NruI-3′ClaI into the “shuttle vector” alreadyencoding for the complete bispecific sc molecule. Alternatively, oligosKC239 and KC240 (splice site only) were annealed and similarly cloned toallow expression of the bispecific sc molecule as a murine kappa lightchain fusion protein.

Example 7 Completion of p149 Bispecific sc Molecule

After cloning the scTCR, Sc-Fv, linker, and tag DNA fragments into the“shuttle vector” to complete the bispecific sc molecule design, the DNAwas restriction digested (AgeI-ClaI) and cloned into the mammalian cellexpression vector pSUN27 (FIG. 5) (previously described in the pendingU.S. application Ser. No. 08/943,086 to create pBISP/149 (FIG. 6).

Example 8 Construction of p149 scTCR/IgG Fusion Molecule

There has been recognition that the expression of the 145-2CII scSc-Fvalone, i.e. not as part of a bispecific sc molecule, is very low.Without wishing to be bound to theory, the low level of sc-Fv expressionmay be a limiting factor in the expression of bispecific molecules.Native 145-2C 11 hybridoma cell line was used as antibody source andcells were transfected with scTCR fused with murine IgG2b heavy chain(FIG. 7A-7B). The transfected hybridoma cell line should secrete some145-2C11/scTCR chimeric molecules if the host's hamster IgG can pairefficiently with murine IgG2b heavy chain.

To clone the p149 scTCR as an IgG fusion, an internal EcoRI restrictionsite was first mutated using site-directed mutagenesis. Briefly, a pairof complimentary oligonucleotides, KC293 and KC294, were designedcontaining the desired mutation. The pNAG2 DNA construct was amplifiedby PCR with the primers using Pfu DNA polymerase. The resulting PCRproduct was digested with DpnI which digests the parental DNA template,leaving the mutated DNA intact. The mutated scTCR DNA was sequenced andthen reamplified by PCR with primers KC276 and KC268 to generate a5′NruI-3′EcoRI DNA fragment. The mutated scTCR DNA was cloned into thepGEM-T Easy Vector System for DNA sequence determination. The correctscTCR DNA was restriction digested and cloned into the mammalian cellexpression vector pSUN7 to create the p149 scTCR/IgG fusion molecule.

Example 9 Construction of DO 11.10 scTCR/IgG Fusion Molecule

The pKC60 DNA construct was reamplified by PCR with primers KC275 andKC268 to generate a 5′NruI-3′EcoRI DNA fragment. The scTCR fragment wascloned into the pGEM-T Easy Vector System for DNA sequencedetermination. The correct scTCR DNA was restriction digested and clonedinto the mammalian cell expression vector pSUN7 to create the DO 11.10scTCR/IgG fusion molecule (See FIGS. 7A and 7B).

Example 10 Construction of the Murine IgG2b Expression Vector

The construction of the murine IgG2b (heavy chain) expression vector wasas follows. The backbone of the vector was the plasmid pcDNA3(Invitrogen). The plasmid was cut with HindIII and XhoI and a “lightchain polylinker” DNA fragment was inserted to create the starting“light chain vector” pcDNA3.LCPL. This linker contained the restrictionsites HindIII, KpnI, ClaI, PmlI, EcoRV, XmaI, BarnHI, and XhoI tofacilitate subsequent cloning steps. A SmaI-BcII DNA fragment containinga light chain leader, mouse anti-CKMB kappa light chain genomicfragment, and 3′ UTR was cloned into the EcoRV-BarnHI sites ofpcDNA3.LCPL. Mutagenesis was then performed to eliminate an NruI MluI,and BstBI site and to introduce an NheI and BarnHI site to create theplasmid pcDNA3mut.LCPL.LCVK.

The “heavy chain vector” pcDNA3mut.HCPL was constructed from thepcDNA3mut.LCPL.LCVK plasmid by replacing the light chain expressionregion (HindIII-XhoI) with a “heavy chain polylinker” consisting ofrestriction sites HpaI, BspEI, EcoRV, KpnI, and XhoI. This plasmid wasdigested with EcoRv and KpnI. A SmaIKpnI digested DNA fragmentcontaining a heavy chain leader and an anti-CKMB IgG2b mouse heavy chaingenomic fragment (see Near et al., Molecular Immun., 1990) was thenligated into the EcoRV-KpnI digested plasmid. A KpnI-SalIoligonucleotide fragment containing a 3′UTR and a NotI site upstream ofthe SalI site was subsequently cloned into the KpnI-XhoI digestedplasmid (knocking out the XhoI site) to create the plasmidpcDNA3mut.HCPL.HCV2b, also known as the murine IgG2b expression vectorpSUN7 (FIG. 8).

Example 11 Expression of Bispecific sc Molecules

CHO cells were prepared for transfection by washing with cold DPBS. Thecells were resuspended in DPBS and mixed with 10-40 ug of PvuIlinearized pBISP/149 or pBISP/DO 11.10. After five minutes on ice, thecells were electroporated using a Gene Pulser (BioRad) set to deliverone pulse of 250 volts, 960μ Fd or 0.25μ Fd. The pulsed cells wereplaced on ice for five minutes. The cells were diluted into 10 ml of 10%IMDM medium (IMDM, 10% FBS, 2 mM glutamine, 5000 units/ml penicillin,5000 ug/ml streptomycin) and grown in a T-25 cm2 TC flask overnight at37 C with 10% CO2 The next day, the cells were plated in 96 well plateswith neomycin selective medium (10% IMDM plus 0.75 mg/ml G418) and refedevery 3-7 days.

Transfectants were screened for expression of soluble bispecific scmolecules in an ELISA assay format. EE-tagged molecules were detectedusing an anti-EE tag antibody passively coated overnight onto a 96 wellplate. On assay day, the plates were blocked with 10% FBS/PBS for onehour. The wells were washed and supernatant from the transfectants wasadded to the plate. After incubating and washing, biotinylated anti-Cbeta mAb H57-597 (cell line was purchased from ATCC) was added to theplate, followed by washing and incubation with streptavidin-HRP (Sigma).Positive wells were identified by the addition of TMB substrate,quenched with 1N sulfuric acid, and read at an absorbance of 450 nM. Asmall number of positive clones were selected for expansion and limitingdilution cloning was carried out to establish stably transfected celllines (FIGS. 9A-9B).

Transfectants were also screened for the expression of bispecific scmolecules in an ELISA assay format using rnAbs which specificallyrecognize the scTCR, followed by detection with biotinylated anti-C betamAb and streptavidin-HRP. For the p149 scTCR bispecific sc molecule, aconformational mAb to the V alpha domain (B20.1, Pharmagen) was used asthe coating antibody. The DO11.10 bispecific sc molecules could bedetected using the anti-idiotypic, anti-DO11.10 TCR mAb KJ-I (FIGS.9A-B, 10A-B). Positive clones were detected as described above, expandedand primary cloned to establish stably transfected cell lines. It hasbeen found that the scBISP molecules are expressed at high levels inmammalian cells (1 to 2 mg/l).

The following information will be helpful in understanding FIGS. 9A-B,10A-B:

FIG. 9A Dilution OD450 1:2 0.4755 neat 0.8545 FIG. 9B: OD450 DilutionH57 B20.1 1:2 0.206 1.21 neat 0.511 1.975 FIG. 10A: OD450 DilutionAnti-EE KJ-1 1:4 0.0825 0.5935 1:2 0.186 0.9095 neat 0.3435 1.1195 FIG.10B: OD450 Dilution Anti-EE KJ-1 F23.1 1:2 0.185 1.143 1.227 neat 0.3811.1655 1.898

Example 12 Expression of Chimeric Bispecific Molecules

The 145-2CI 1 hybridoma cell line was transfected with either p149scTCR/IgG fusion DNA or DO11.10 scTCR/IgG fusion DNA using the samemethod as described above for the bispecific sc molecule transfection.

Transfectants were screened for expression of soluble chimericbispecific molecules in an ELISA assay format. 96 well plates werepassively coated with goat anti-mouse IgG2b (Caltech). Incubation andwashing steps were performed as described above. Goat anti-hamsterIgG-HRP (Jackson Immuno.) was used to probe the wells (FIG. 11).Positive colonies were identified, expanded and primary cloned toestablish stably transfected cell lines.

The following information will be helpful in understanding FIG. 11:

OD450 Construct neat 1:2 BISP/149 0.6305 0.2985 BISP/DO1 0.964 0.6983

Example 13 Purification of Bispecific sc Protein

Bispecific sc proteins were purified from transfectant supernatant usingstandard affinity chromatography methods. For EE-tagged proteins, ananti-EE tag CNBr-coupled agarose column was used to enrich forfull-length sc molecules. Supernatant was passed over the column bed oneor more times. After washing with PBS, the bound protein was eluted offthe column by the addition of high pH sodium bicarbonate/carbonatebuffer and neutralized by the addition of a 1 to 10 dilution of 2M Tris,pH8.0. The purified protein was buffer exchanged into PBS using a 30 kDMW cut-off concentration unit. The final protein concentration wasdetermined by an OD280 reading. Western blot analysis (probed withanti-EE tag antibody) (FIG. 12) and coomassie-blue staining of thepurified protein (FIG. 13) show enrichment for the full-lengthbispecific sc molecule.

Example 14 Bispecific Sc Molecule Stimulation of T Hybridoma Cells

T hybridoma cell stimulation assays were performed to assess whether thebispecific sc molecules displayed biological activity. We developed aworking model system using the murine T cell hybridoma 2B4 (Matsui, etal., PNAS USA. (1994) 91, 12862) The 2B4 T cell hybridoma has an/13 TCRconsisting of V 11.0 and Vβ3.0 and recognizes amino acid residues 88-104of pigeon cytochrome C presented in the context of MHC class II moleculeIE^(k) Several different immobilized Abs specific for either the DO11.10or 149 TCR were tested for cross-reactivity to 2B4 TCR, but turned outto be unreactive towards the 2B4 TCR. If an immobilized Ab demonstratedcross-reactivity for the 2B4 TCR, we would expect to observe stimulationof the T hybridoma cells and secretion of L-2 into the culturesupernatant. The Abs evaluated included two specific for 149 TCR, theanti-V 2 and anti-Vβ11, and two specific for the DO11.10 TCR, theanti-Vβ8.0 (F23.1) and the anti-idiotypic mAb (KJ-1). Also, we evaluatedimmobilized IA^(d)/OVA (the cognate MHC/peptide for the DO11.10 TCR),but did not observe any stimulation. We then immobilized these moleculesand evaluated the activity of the DO11.10 and 149 bispecific scmolecules. To test the DO11.10-2C11 bisp. sc molecule, we coated wellswith either KJ-1 or F23.1. After incubation overnight with 10⁵ 2B4 cellsusing different amounts of bispecific, we assayed supernatant for thepresence of IL-2 which is a good indicator of cell stimulation. As shownin FIG. 13, immobilized KJ-1 effectively activated the hybridoma cells.To evaluate whether a similar response could occur when usingimmobilized IA^(d)/OVA, we next incubated the 2B4 cells with bispecificmolecules overnight in the presence of plate-bound IA^(d)/OVA. Thepresence of IL-2 in the supernatant was not detected in the IL-2 ELISAassay (FIG. 14) suggesting the TCR on the bispecific was not engagingthe MHC/peptide ligand in a manner sufficient for cell stimulation. Weproposed based on these findings and several others that it may beessential to improve the avidity of the bispecific sc molecules throughdimerization using an antibody specific for the TCR but would notinterfere with the bispecific binding to MHC/peptide. In our example, wechose the MR5-2 mAb (PharMingen) which has specificity for an epitope onVβ8.2. After assaying under these modified conditions, we observed asignal in the wells containing immobilized IA^(d)/OVA but did not detecta signal in blank wells. Furthermore, in wells coated with KJ-1 mAb, theeffect of cross-linking of the bispecific sc molecule generated a higherIL-2 output suggesting dimerization of the bispecific sc molecule leadsto perhaps a stronger and/or different signaling and stimulation (FIG.15).

The following information will be helpful in understanding FIGS. 14 and15:

FIG. 14 BISP ng/well OD450  2 0.013  3.9 0.036  7.8 0.196  15.6 0.72 31.2 1.01  62.5 1.3375 125 1.746 250 2.066 FIG. 15: Construct BISPBISP + MR5 Blank 0 0.12 IAd/OVA 0.01 0.271

To evaluate the 149-2C 11-EE-tag bispecific sc molecule, we coated wellswith either anti-V 2.0 or anti-Vβ11.0 mAb. Blank wells were used asnaive controls. The findings generated in these experiments were similarto those reported for the DO 11.10-2C 11 bispecific-sc molecules andshowed that only in the presence of immobilized Ab specific for the 149TCR did we observe IL-2 production (FIG. 16). Furthermore, in thisexample, we demonstrated that the cross-linking by the bispecific tostimulate the T hybridomas could be effectively blocked using solubleanti-CD3 F(ab)′₂ 145-2C 11. These findings argue favorably forfunctional bispecific sc molecules and show the anti-CD3 portion of thebispecific acts by binding directly to the CD3 molecule on T cells (FIG.16).

The following information will be helpful in understanding FIG. 16:

OD450 Construct 1:2 1:4 1:2/CD3 1:4/CD3 Blank 0.01 0.01 0.01 0.01 RR3-151.07 1.03 0.185 0.282 B20.1 1.2 1.08 0.112 0.118

Example 15 Flow Cytometric Analysis for Direct Cell Binding Studies

To demonstrate functionality of the scSc-Fv portion of the bispecific scmolecule, 2B4 T hybridoma cells were used in binding studies with thepurified protein. 2B4 cells display CD3 on their surface and correctlyfolded 145-2C11 sc-Fv should recognize CD3ε. For each test sample, 10⁶2B4 cells were washed with cold DPBS and resuspended in 40ul of 1%FBS/DPBS (resuspension and washing buffer) with or without the additionof purified bispecific sc protein. After incubation on ice, the cellswere spun down gently and resuspended with 0.5 ug of biotinylatedantibody (pBISP/149 was incubated with an antibody to the Va2 domain(B20.1); pBISP/DO11.10 was incubated with an antibody to the Vβ8 domain(F23.1).) Samples were incubated on ice, spun down, and resuspended withstreptavidin-cychrome (Becton Dickenson). After washing two times, thecells were resuspended again and then acquired/analyzed on a FACScaninstrument (Becton Dickenson) using CellQuest software (BectonDickenson).

Incubation of 2B4 cells with either the pBISP/149 or pBISP/DO11.10purified protein resulted in significant shifts in cell staining. Asmore bispecific sc protein was added, the shift in fluorescence was morepronounced, demonstrating the ability of the scSc-Fv to bind to the CD3on the cell surface (FIGS. 17-18).

The CD3 binding is specific and can be blocked by the addition ofsoluble anti-CD3 which competes with the bispecific sc molecules forbinding sites on the 2B4 cell surface (FIG. 19).

FIGS. 17-19 are more fully understood in light of the following TablesI, II and III below.

TABLE 1 [FIG. 17] Key Name Parameter Gate — 062498.001 FL3-H No Gate —062498.002 FL3-H No Gate — 062498.003 FL3-H No Gate — 062498.004 FL3-HNo Gate Marker Events % Gated % Total Mean Median Peak Ch File:062498.001 [No BISP] Sample ID: 2B4 ANTI-VA2- B-SA-CY Gate: No Gate All10000 100.00 100.00 7.78 3.19 1 File: 062498.002 [1X BISP] Sample ID:2B4 1UL149 BISP ANTI VA2-B-SA-CY Gate: No Gate All 10000 100.00 100.00.10.96 7.10 8 File: 062498.003 [10X BISP] Sample ID: 2B4 10UL 149BISPANTI-VA2-B-SA-CY Gate: No Gate All 10000 100.00 100.00 88.96 62.08 55File: 062498.004 [25X BISP] Sample ID: 2B4 25UL 149BISP ANTI-VA2-B-SA-CYGate: No Gate All 10000 100.00 100.00 186.06 177.83 215

TABLE 2 [FIG. 18] Key Name Parameter Gate — 062498.009 FL3-H No Gate —062498.010 FL3-H No Gate — 062498.011 FL3-H No Gate — 062498.012 FL3-HNo Gate Marker Events % Gated % Total Mean Median Peak Ch File:062498.009 [No BISP] Sample ID: 2B4 ANTI-VB8.2-B-SA-CY Gate: No Gate All10000 100.00 100.00 8.08 2.48 1 File: 062498.010 [1X BISP]Sample ID: 2B41UL DO11BISP ANTI-VB8.2-B SA-CY Gate: No Gate All 10000 100.00 100.007.51 3.65 3 File: 062498.011[5XBISP] Sample ID: 2B4 5UL DO11BISPANTI-VB8.2-B SA-CY Gate: No Gate All 10000 100.00 100.00 19.31 14.46 15File: 062498.012 [10XBISP]Sample ID: 2B4 10UL DO11BISP ANTI-VB8.2-BSA-CY Gate: No Gate All 10000 100.00 100.00 28.29 24.56 27

TABLE 3 [FIG. 19] Key Name Parameter Gate — 051398.003 FL3-H G1 —051398.002 FL3-H G1 — 051398.005 FL3-H G1 — 051398.006 FL3-H G1 Events %Gated % Total Mean Median Peak Ch File: 051398.003 [No BISP/No αC3]Sample ID: 2B4 VA2 CYCH PI Gate: G1 Gated events: 9853 Total Events:11724 9853 100.00 84.04 5.20 4.66 4 File: 051398.002 [BISP, NoαCD3]Sample ID: 2B4 BISP VA2 CYCH PI Gate: G1 Gated events: 9907 TotalEvents: 11420 9907 100.00 86.75 8.21 7.84 9 File: 051398.005 [No BISP,No αCD3] Sample ID: 2B4 ANTI-CD3 VA2 CYCH PI Gate: G1 Gated events: 9905Total Events: 11715 9905 100.00 84.65 5.60 5.14 4 File: 051398.006[BISP, No αCD3] Sample ID: 2B4 ANTI-CD3 BISP VA2 CYCH PI Gate: G1 Gatedevents: 9933 Total Events: 11218 9933 100.00 88.55 5.43 5.00 5

Example 16 T Cell Proliferation Assay

A T-cell assay was performed to determine whether the scBisp 149molecule could mediate specific T cell activation. A proliferation assaywas carried out using long-term cultured T cells, cultured in thepresence of unpulsed or 149 peptide pulsed T2 (29) target cells that hadbeen fixed in 1% paraformaldehyde prior to being used in the assay.Conditions were chosen to test whether the scBisp 149 molecule couldactivate T cells to proliferate when incubated with unpulsed or p149peptide pulsed T2 target cells. The assay was carried out as follows.Briefly, spleens isolated from BALB/c mice were used to preparesplenocyte suspensions. RBCs were lysed using Gey's solution and therecovered splenocytes were then cultured for 10-15 days at 1.25×10⁶cells/mL in IMAM media supplemented with 10% Fetal Bovine Serum (FBI)containing 50 U/mL of murine rIL-2. Media was changed every 3 days andnon-adherent cells were recovered, counted and resuspended at 1.25×10⁶cells/ml. Before using the cells in the proliferation assay, live cellswere isolated on a Ficoll-Hypaque density gradient. The cultures wereincubated for 3 days and T cell proliferation was measured using thecolorimetric proliferation reagent WST-1 (Boehringer Manheim) accordingto the manufacturer's instructions. As shown in FIG. 20, only T cellsincubated in the presence of the bispecific and the 149 peptide loadedT2 cells demonstrated significant proliferation, whereas the culturesincubated in the absence of either the 149 peptide or the scBishp 149molecule did not exhibit proliferation. These data were significantbecause they illustrate “proof of principle” that scTCR used in a hybridscBisp molecule format can mediate T cell responses to target cellspresenting HLA-A2 and the specific peptide.

Spleenocytes were prepared from spleens isolated from Balb/c mice.Briefly, RBCs were removed by lysing using Gey's solution and therecovered spleenocytes were then cultured for 10-15 days at 1.25×10⁶cells/ml containing 50 U/ml of murine rIL-2. Media was changed every 3days and non-adherent cells were recovered, counted and resuspended at1.25×10⁶ cells/ml. Before using the cells in the proliferation assay, weisolated the live cells (primarily T cells) on a Ficoll-Hypaque densitygradient. In this example, we tested whether the 149-2C11 sc moleculecould effectively recognize and bind to cognate MHC/peptide onpresenting cells and facilitate cross-linking and activation of T cells.The proliferation assay was carried out using long-term cultured Tcells, cultured in the presence of unpulsed or 149 peptide pulsed T2target cells that were than fixed in 1% paraformaldehyde prior to beingused in the assay. The cultures were incubated for 3 days and T cellproliferation was measured using the colorimetric proliferation reagentWST-1 (Borhringer Manheim) according to the manufacturer's instructions.After a 1 hour incubation at 37° C. 100 μl of supernatant wastransferred to a flat bottom plate for reading at dual wavelength(450-620 nary). As shown in FIG. 19, T cells incubated in the presenceof the bispecific and the 149 peptide loaded T2 cells demonstratedsignificant proliferation, whereas the cultures incubated in the absenceof p149 peptide or bispecific did not exhibit any significantproliferation. These data support the T-hybridoma stimulation resultsdescribed above and suggest the 149-2CI I bispecific sc molecule isbiologically active.

Example 17 Profiling Cytokine Production

Another important parameter to evaluate is the ability of the bispecificsc molecule to mediate cytokine responses. Cytokine production can bedetected by an ELISA assay specific for the cytokine of interest. 96well plates are passively coated with anti-cytokine “A” overnight. Onassay day, the wells are blocked with 10% FBS/PBS for one hour beforeadding supernatant from the proliferation-type experiment. Wells areprobed with biotinylated anti-cytokine “A” followed by incubation withstreptavidin-HRP. Positive wells are detected by the addition of ABTSsubstrate and read at an absorbance of 405 nM

Cytokine production can also be looked at intracellularly using asaponin permeabilization protocol. The cells are fixed with formaldehydeand then stained for the cytokine of interest in the presence of 0.5%saponin. Samples can then be analyzed using flow cytometry.

Example 18 Measuring In Vitro Cytotoxic Activity

One-of the most important parameters to measure will be whether thebispecific sc molecule can mediate target or tumor cell killing. Theseassays will be carried out using a standard Cr⁵¹ release CTL killingassay. The assay will be run as follows: Target cells (i.e. tumors) arefirst labeled with the isotope Cr⁵¹. The Cr⁵¹ is taken up by the tumorcells and is released into the culture supernatant upon cell lysis bythe specifically activated cytotoxic T cells. The free or released Cr⁵¹is then counted and the specific cell lysis determined. We will use thistype of an assay to evaluate the 149-2C11 sc molecule's ability tomediate target cell lysis. In our assay, we will use tumor cell lines(i.e. MDA-238, BT549, and MCF-7 available from ATCC) known to expresssurface HLA-A2 and produce increased levels of wild-type p53. Controlswill include A2 negative tumor lines (Ramos) and A2 positive but p53negative cell lines (Saos-2).

Example 19 Generation of Bispecific Molecules Through ChemicalCross-Linking to Dendrimers

To construct the bispecific molecule using a chemical cross-linkingapproach, the 2C11 mAb and the DO11.10 scTCR was used. Instead ofdirectly cross-linking the two molecules, dendrimers were used as ascaffold to attach the molecules. Dendrimers are positively chargedpolyamines that are uniformly synthesized. Because the size and shape ofeach dendrimer derived during synthesis is exactly the same, theaddition of proteins to the dendrimer results in the formation ofhomogenous molecules. The dendrimer also is inert and soluble underphysiological conditions. Full-length 2C11 mAb was pepsin digested toproduce F(ab)′2 fragments which were isolated by gel filtration. TheF(ab′)₂ peak was pooled and buffered exchanged and Fab′ molecules wereproduced by incubating the F(ab′)₂ preparation under mild reducingconditions followed by purification on a sizing column. The isolatedFab′ molecules were then directly coupled through free sulfhydryl groupsto sulfo-succinimdyl (4-iodoacetyl) amino benzoate (sulfo-SIAB)derivatized dendrimers at a ratio of one Fab′ to one SIAB derivatizeddendrimer. Reactive-aldehyde groups were generated on terminalcarbohydrate residues of the D011.10 scTCR molecule for coupling to freeamine groups on dendrimers. The scTCR was coupled to the 2C11 Fab′dendrimer at a 1:1 ratio to yield a bispecific molecule. The bispecificmolecule was evaluated for its ability to activate T hybridoma cells ina stimulation assay. A working model system was developed using themurine T cell hybridoma 2B4. See Davis, M. M. et al. Ciba Fund. Synp.(1997). The 2B4 T cell hybridoma has an/TCR consisting of V 11.0 andVβ33.0 and recognizes amino acid residues 88-104 of pigeon cytochrome Cpresented in the context of MHC class II molecule IE^(k). When thebispecific/dendrimer was added to wells containing 2B4 T cells highlevels of IL-2 were reported indicating stimulation had occurred.Further analysis revealed that the strong positive charge on thedendrimer complex caused non-specific binding to the T cell surfaceresulting in stimulation.

Example 20 Mouse Models: Evaluation of the Bispecific Molecule'sActivity In Vivo

Three different established murine models have been established in orderto evaluate the potential tumor suppression activity of the bispecificmolecules. The first model includes using a normal mouse strain (i.e.Balb/c mouse) and injecting into this mouse pS3/HLA-A2 transformed EL-4cells. These tumor cells proliferate quickly and within a few days killthe mouse. The following treatment protocol will be initially used. Toevaluate our bispecific molecule, mice will be pre-treated with 0.5 mgof bispecific 1 49-2C11 sc molecule on day 0. The following day the micewill receive a second dose of the bispecific sc molecule along with thep53/A2 positive transfected EL-4 cells. The main parameter to measure inthis model will be whether mice that receive the bispecific moleculesurvive for a longer period of time than control mice (ones that did notreceive the bispecific molecule). Because the EL-4 tumor lines displayssuch rapid growth, we may be required to modify the treatment regimenfor us to observe any increased survival time with the bispecific scmolecule.

The second model will use SCID mice implanted with murine tumorstransfected to express HLA-A2 and human wild-type p53. This modelusually runs for two to three weeks. Briefly, after implanting tumors,we can measure the growth of the tumor and then introduce into thesemice purified murine CD8+ T cells and inject different amounts of thebispecific molecule. In some cases, we will need to pre-activate the Tcells and this will be carried out by incubating T cells in vitro in thepresence of rIL-2. We will then evaluate the affect on tumor growth andthe change in survival time to determine whether the bispecific scmolecule has anti-tumor activity in vivo.

The third model and most relevant will evaluate the ability of thebispecific sc molecule to mediate tumor killing in in vivo of humantumors. SCID mice will be implanted with human breast carcinoma lines(i.e. MDA-238, BT549, MCf-7) and allowed to grow for 4 to 6 weeks. Thenpurified T cells and subset populations pre-activated in in vivo withrIL-2, will be introduced into the mice. The potential anti-tumoractivity will be assessed by measuring tumor reduction and increasedsurvival time. These studies will be used to determine whether a“humanized” version of the bispecific molecule should be constructed.

Example 21 “Humanized” Bispecific sc Molecule

Because the antibody used in our current bispecific sc molecule isspecific for murine CD3ε, we will have to modify it for use in treatinghuman neoplasms. Furthermore, if we use hybridoma technology, we willmost likely isolate murine mAbs specific to human TCRs or CD3 that willhave to undergo “humanization”. The humanization will be earned outdoing CDR grafting. This usually has the negative affect of decreasingthe binding avidity of the Ab. The TCR can be “humanized” primarilythrough swapping out the C beta constant domain with the human C betaconstant region.

Example 22 Display of sc-TCR Fusion Proteins on Bacteriophage

As disclosed in the pending U.S. application Ser. No. 08/813,781, it ispossible to display a variety of sc-TCR constructs on the surface of fdbacteriophage. Briefly, the pending application discloses methods ofexpressing a desired three domain sc-TCR as a fusion with the major coatprotein, pVIII, of filamentous phage. The rationale for this is toincrease the valency of the scTCR on the surface of the phage whichshould result in an increase in the avidity of scTCR/pVIII for theMHC/peptide complex. As disclosed in the pending U.S. application Ser.No. 08/813,781, the sc-TCR fusion proteins on the bacteriophage displaya functional TCR.

A. Characterization of Displayed sc-TCR Fusion Protein

1. Western Blot Data

Many studies have been published showing scFv/pVIII fusion proteinsexpressed on the surface of phage. See Castagnoli et al., J. Mol. Biol.,(1991), 222: 301 and Huset et al., J. Immunol., (1992) 149:2914. Thepending U.S. application Ser. No. 08/813,781 discloses methods of makingand using specific recombinant bacteriophages that display sc-TCR fusionproteins. Here, Western blot analysis was used to confirm display of thescTCR/pVIII molecule on the capsid coat of the phage. Briefly,bacteriophage were purified by means of a standard polyethylene glycol(PEG) precipitation procedure, and subsequently an aliquot of thepurified phage was run on an SDS-PAGE gel. The scTCR/pVIII fusion wasdetected in the recombinant phage (but not in control phage expressingscTCR without the EE-tag) by probing the membranes with a mAb againstthe EE tag sequence. Although several smaller bands representingbreakdown products of the scTCR fusion were observed, the presence of a50 kD protein band indicated a full length α/β scTCR had beenincorporated into the phage capsid.

2. ELISA data

As disclosed in the pending U.S. application Ser. No. 08/813,781, ELISAassays can be used to characterize recombinant bacteriophage thatinclude a desired sc-TCR fusion protein. The conformational integrity ofthe Vβ8.2 chain was evaluated using two conformational dependent mAbs,MR5-2 and F23.1; and the precise folding of the Vα13.1, Jα D0, Vβ8.2,Dβ1, and Jβ1.1 domains which form the CDR3 binding pocket of thereceptor was assessed using the anti-idiotype mAb KJ1. Background signalwas considered as phage binding to wells coated with either BSA or mAbanti-Vβ17 and was subtracted from the total signal observed. The fourantibodies reacted specifically with the phage TCR indicating thescTCR/pVIII fusion was presented on the phage in the proper orientation.

B. Phage Panning

Panning of antibody and peptide libraries is firmly established as amethod to reliably screen for specific binding molecules, Greenwood,supra. Methods for panning bacteriophage that display sc-TCR fusionproteins have been disclosed in the pending U.S. application Ser. No.08/813,781. Briefly, the methods include standard antibody, cellpanning, and panning with sc-MHC/peptide complexes disclosed inpublished PCT Application No. US 95/09816 as well as the pending U.S.application Ser. Nos. 08/382,454 and 08/596,387. Results from theseenrichment studies correlate well with other published antibody panningfindings. See, Winter et al., Annu. Rev. Immunol., (1994), 12.

C. Blocking Assay

To characterize the MHC/peptide binding specificity of the TCR bearingphage, a competitive blocking assay. The competitive blocking assay hasbeen disclosed in the pending U.S. application Ser. No. 08/943,086.Briefly, the objective of this example was to determine whether TCRcarrying phage could compete with the native TCR on DO11.10 hybridoma Tcells for binding to MHC/peptide complexes in a cell based assay. Theresults demonstrate the DO11.10 receptor on phage was functional and wasable to discriminate between different peptide sequences.

To eliminate the possibility that the TCR carrying phage had perhapsaffected the IL-2 production of the DO11.10 cells in a non-specificmanner, wells were coated with mAb anti-CD3 epsilon to stimulate thehybridomas through the T cell receptor complex CD3 molecule to produceIL-2. Results from these experiments indicate that the phage did nothave a non-specific inhibitory effect on the T hybridoma cells. Thus,the scTCRs are displayed on the surface of bacteriophage as functionalmolecules which are able to interact with specific MHC/peptide targets.

Example 23 Cloning and Expression of the F23.1 scFv

A preferred component of the polyspecific binding molecules disclosedherein is a an scFv with specificity for a particular sub-population ofT cell receptors. As discussed a wide spectrum of different sc-Fvmolecules can be used in accord with the present invention.

A more specific polyspecific binding molecule is a bispecific moleculewhich includes a single-chain form of the murine mAb F23.1. It ispossible to clone and express such a sc-Fv by standard techniques. Thenative F23.1 antibody has been well characterized (1) and has been shownto activate Vβ8.2 bearing T cells by cross-linking the TCR on itssurface, Hiller et al., Biochem. J., (1991) 278: 573. The sc-Fv can becloned and expressed by the following general steps.

1. cDNA synthesis and cloning of the heavy and light chain genes ofF23.1

First strand cDNA synthesis can be accomplished with mRNA isolated from107 F23.1 cells. Using primer JS300 (GAAX₁TAX₂CCCTTGACCAGGC whereinX₁=A,G and X₂=A, C, G; SEQ ID NO. 12), we synthesized heavy chain cDNA.This primer encodes for the first two amino acids of the heavy chain CH1domain. The light chain cDNA was synthesized essentially the same wayexcept we used primer OKA57 (GCACCTCCAGATGTTAACTGCTC; SEQ ID NO. 13)which is specific for the 3′ end of framework four of the kappa chain.

Double stranded DNA was made by amplifying the cDNA as follows. Heavychain was amplified by using primer set PMC18 (front)(CCCGGGCCACCATGGX₁ATGX₂AGCTGX₃GTX₄ATX₅CTC; wherein X₁=A,G; X₂=C,G;X₃=G,T; X₄=A,C; X₅=C,G; SEQ ID. NO. 14 and JS300 and the light chain wasamplified using primer set PMC14 (front)(CCCGGGCCACCATGGAGX₁CACAX₂X₃CTCAGGTC, wherein X₁ and X₃ are and X₂ isG,T; SEQ ID NO. 44) and OKA57. The amplified PCR products were thencloned into pGEM T-easy vector (Promega) and submitted for nucleotidesequence determination. A subsequent step will be to clone the heavy andlight chains into a single-chain format for expression of scFvfragments.

Example 24 Cloning and Expression of the F23.1 Antibody as aSingle-Chain Molecule

It is possible to clone a single-chain version of the F23.1 antibodywhich has been shown to recognize a conformational determinant on murineT cells bearing Vβ8.2 TCRs. In general, the Vβ8.2 family of TCRs isexpressed at a frequency of 20% on T cells in most strains of mice,Staertz, U., J. Immunol., (1995) 134, 3994. After the antibody gene hasbeen cloned it will be possible to express the scFv molecule in E. colifor characterization.

The full length heavy and light chains (i.e. V/CH; V/CL) representingthe F23.1 antibody have been cloned separately into vector pGEMT-easy(Promega) and the VH and VL genes have been confirmed by sequencing. TheF23.1 antibody gene will be cloned into a single-chain molecule bysplicing together the genes encoding the VH and VL domains. For example,one approach is to clone the scFv into the expression vector pEN2, forproduction of scFv fragments in E. coli. The pEN2 vector has beendisclosed e.g., in U.S. Pat. No. 5,763,284.

The cloning protocol can be performed by using a two-step PCRamplification process. In the first round, the VH and VL genes will beseparately amplified by using a specific primer set that anneals to the“front” and “back” of the VH and VL genes. In future experiments, Tcells will be targeted to tumors, using as the antibody portion,antibodies reactive to CD3, CD4, and to particular TCRs. However, thisparticular antibody is preferred, e.g., because it can activate T cells,and more specifically activate CTLs. To make the scFv construct, asecond step amplification will be carried out using overlapping PCRmethodology to “splice” together the VH and VL genes. The two chains arelinked using a 16 amino acid linker (G4SG4APG4S) containing therestriction site for the 8 base cutter AcsI. In those instances whereoverlapping PCR must be undertaken, two primers can be used as follows:JSS32(T) (GGTGGCGGCGCGCCGGGAGGCGGCGGTTC; SEQ ID NO. 15) which overlapswithin the linker sequence on the 5′ end of the light chain and thebottom primer JSS33(B) (GCCTCCCGGCGCGCCGCCACCACCGCTGCCACCGCCACC; SEQ IDNO. 16) which overlaps within the linker region and runs into the 3′ endof the heavy chain. The overlap PCR product is digested with SfiI andSpeI and then isolated by running the sample on a 1% agarose gel andexcising the scFv band.

The scFv gene is then cloned into the expression vector pEN2 as a Sfi/toSpeI fragment and induction of the protein is controlled by the phoApromoter. It is believed that approximately 50% of the scFv moleculescan be expressed in E. coli as soluble and functional protein. If aparticular host cell or culturing condition produce insoluble protein,the sc-Fv can be refolded according to standard techniques to obtainsoluble protein.

Example 25 Construction of scFv/pIII Fusions for Expression onBacteriophage

As discussed, it is possible to express a variety of sc-Fv fusionproteins on the surface of a bacteriophage as one component of the phagecapsid. For example, it is possible to make a bispecific bacteriophageendowed with binding affinity for an epitope on a desired T cellreceptor and on a target tumor cell.

More specifically, to make an example of the bispecific phage, the F23.1antibody molecule will be cloned as a scFv/geneIII fusion. As discussedin the pending U.S. application Ser. No. 08/813,781, the geneIII proteinis a 406 amino acid protein that is expressed as five copies on thesurface of phage. An specific fd tet bacteriophage has been modified byadding convenient SpeI and NotI sites for cloning scFv genes as pillfusions. By amplifying the F23.1 scFv gene from the pEN2 vectordescribed above in Example 23, the gene will be cloned as an SpeI-NotIfragment into the N-terminal region of the wild-type pIII protein.Because induction of the scFv/pIII fusion is under the control of thetac promoter, expression will occur after the addition of 1 mM of IPTG.Generally, it has been reported that one copy of the scFv is displayedper phage as a gene III fusion. Therefore, the phage represent anattractive system to use for displaying single copies of the antibodymolecule. This will help to minimize non-specific interactions and willallow the phage to closely mimic the ideal concept of displayingmonovalent scFv molecules. See FIGS. 2D-2E for examples of specificsc-TCR fusion constructs.

1. Modifications to the scFv Construct by Adding Specific Tags

Several alternate ways will be used to make scFv molecules containingcarboxyl terminal region tags. DNA encoding for the sequence of each tagwill be fused to the 3′ end of the light chain gene. The inclusion of acarboxyl terminal tag will result in detection of the molecule. Examplesof tags include the KT3, TPPPEPET; (SEQ ID NO. 10,); 6×His, GMAHHHHHH;(SEQ ID NO. 9,) avidin; ARKCSLTGKWTNDLGSNMT; (SEQ ID NO. 6) fos, BirA,LXLIFEAQKIEWR (SEQ ID NO. 5) or jun. The molecule KT3EE (EEEEYMPME, SEQID NO. 8) may also be used in some instances. See Hiller et al., (1991),Biochem., J., 278, 573; Patel et al., 1994), Proc. Natl. Acad. Sci. USA,(1994), 91, 7360; Kruif et al., J. Biol. Chem., 271: 7630 and Schatz,P., Bio/Technology, (1993) 11, 1138.

Example 26 Purification and Analyses of scFv F23.1 for Binding to Nativeand Single-Chain TCR

Metal-chelating chromatography has become a widely used procedure in thepurification of recombinant proteins. This technology can be used topurify the scFv molecule. A 6×His tag has been engineered into thedesign of the scFv molecule to allow simple purification on a Ni2+NTAcolumn. Preparation of the soluble fraction is accomplished bysuspending E. coli cell paste in extraction buffer and cells are thenlysed in a French press. The sample will then be applied to a Ni2+NTAcolumn under conditions that allow for binding of 6×His tagged proteinsand bound protein will be eluted using imidazole, pH 7.4. Samples willbe analyzed for purity by SDS-PAGE and coomassie blue staining of thegel. Western blot analysis will be used to evaluate the integrity of thescFv by probing membranes with an antibody specific for a KT3 tag.

If desired, additional purification of the sc-Fv may be performed asfollows. For example, an affinity column can be made by first covalentlycoupling the anti-EE tag mAb to protein-A sepharose beads. The anti-EEtag coated beads will then be used to capture the purified D011.10 scTCRwhich will then be cross-linked to the mAb. The column can be used topurify the scFv (F23.1) antibody because it has specificity for theVβ8.2 domain. This two-step purification scheme will yield a homogeneousscFv preparation.

To assess whether the purified scFv F23.1 is functional, plasmon surfaceresonance studies can be used to measure the binding constantassociations of the scFv to scTCR(DO11.10) protein biotinylated andcoupled to a streptavidin coated chip. As a control, the bindingconstant association of the native F23.1 antibody can be determined forcomparison with the recombinant F23.1 antibody. See the pending U.S.application Ser. No. 08/813,781 for disclosure relating to performingthis assay.

In addition to comparing the binding profiles between the native andrecombinant F23.1 antibodies for the scTCR, it is possible toinvestigate the ability of these antibodies to bind to the native TCR onDO11.10 T cells. Flow cytometric analysis will be employed to detectbinding of biotin labeled antibodies to the receptors on the cells. Itis anticipated that the data from these experiments will predict theability of the scFv fragment to activate T cells through binding of theVβ8.2 bearing receptors. Disclosure relating to performing flowcytometric analysis can be found in the pending U.S. application Ser.Nos. 08/813,781 and 08/943,086.

Example 27 Expression of the F23.1 scFv as a geneIII Fusion on theSurface of Bacteriophage

As discussed, the F23.1 sc-Fv can be expressed as a pIII fusion fordisplay on the surface of bacteriophage. This approach represents aunique way to rapidly characterize the scFv molecule before making theeffort to express and purify the antibody from E. coli. To accomplishmany of the characterization studies described, sufficient numbers ofphage can be obtained from 2- to 5 liters of an overnight culture.Moreover, as an alternative to purying the sc-Fv molecules as describedin Example 6 (instead of using an elaborate affinity purification systemas we have described for purifying scFv molecules from E. coli lysate),the purification of the bacteriophage can be easily accomplished by tworounds of polyethylene glycol (PEG) precipitation. If it is necessary tofurther purify the phage, enrichment on a CsCl gradient can be used.After cloning, the phage expressing the scFv can be produced, purifiedand available for testing within a shorter period of time compared toexpression and purification of soluble scFv molecules. Cell bindingassays can be used to test whether phage are expressing scFv/pIIIfusions. This will be carried out by incubating phage with DO11.10 Tcells and assaying for, bound phage using a biotin labeled antibodyspecific for phage. More specific disclosure relating to performing thecell binding assays can be found in the pending U.S. application Ser.No. 08/813,781. Detection of antibody labeled phage will be analyzed byadding a streptavidin-phycoerythrin (PE) conjugate. scFv moleculesexpressed as pill fusions normally retain conformational activity and inmany instances perform better than the recombinant Fv fragment. One goalis to confirm that a functional scFv fragment is displayed only on thetip of the phage.

Example 28 Ability of Recombinant Bacteriophage to Activate T Cells

The F23.1 sc-Fv molecule preferably exhibits an ability to stimulateDO11.10 T cell hybridomas or to activate a population of non-enrichedmurine T cells. All experiments will use the native F23.1 antibody as apositive control, especially since reports have shown it can activate Tcells. See Staerz, supra. In the first experiment, bacteriophageexpressing the scFv/fusion will be adsorbed directly to the plastic wellor immobilized by capturing with an antibody to the phage. Anotherapproach will use the phage in suspension. Briefly, 105 DO11.10 Thybridoma cells will be added to wells containing phage as describedabove. After an overnight incubation, plates will be centrifuged topellet cells and the supernatant isolated and assayed for IL-2 using ananti-IL-2 ELISA.

The second experiment will use T cells isolated from Balb/c mice. Twoparameters will be measured to evaluate activation. The first parameterwill be to assay IL-2 levels after incubating 106 naive cells in thepresence of the antibody phage. The second method will be to stain naivecells for activation markers identified on T cells after incubation withthe phage. It is possible to assay a number of surface markers that areexpressed or up-regulated after activation. Parallel studies can beperformed using the purified scFv instead of the phage. However, in thiscase it is preferred that the molecule will be immobilized with an mAbhaving specificity for the KT3 tag located at the carboxyl terminalregion of the scFv molecule. From these two experiments, it is possibleto evaluate the effectiveness of using scFv phage to stimulate oractivate T cells.

Example 29 Engineering Bispecific Hybrid Molecules (i.e. BispecificBacteriophage) and Characterization of its Tumor Killing Properties InVitro

One objective of the present invention is to illustrate that the presentpolyspecific binding molecules and particularly the sc-TCR/sc-Fvbispecific molecules can kill tumor cells in vitro. The bispecificmolecules can be made in a number of ways in accord with the inventionincluding the following specific methods.

One approach to making bispecific molecules will be to utilizebacteriophage as a vehicle for displaying simultaneously both the scTCRand the scFv. Bispecific phage will be made by infecting K91 E. colicells previously transformed with the pSUN26 phagemid vector, and thenwith scFv/pIII expressing phage. After PEG purification of thebispecific phage, the phage can be characterized to ensure both scTCRand scFv fusions are expressed on the surface. This will be done byELISA assay in which wells will be coated at lug/well of purified scTCR(DO11.10) bearing the V 8.2 epitope. Phage that express a functionalscFv F23.1 should bind to the DO11.10 scTCR. Streptavidin-HRP will beused to detect the p-149 scTCR displayed on the surface of phage. Anti-V2.3 or anti-V 11.0 mAb (PharMingen) will be used followed bystreptavidin-HRP.

Example 30 Strategies for Linking scTCR and scFv Molecules with JoiningMolecules

As discussed, the present polyspecific binding molecules can be joinedby one or a combination of different joining molecules. Several specificjoining molecules include immunoglobulin heavy chains and the moleculesdisclosed above that are capable of forming specific binding complexes.

For example, one specific type of joining molecule is the biotin bindingmotif of avidin. This joining molecule has been genetically encoded intothe design of the p-149 scFv molecule. Therefore, scFv molecules will beexpressed with a sequence of amino acids derived from avidin whichcontains a biotin binding motif (See, Hiller et al., Biochem., (1991),278, 573-585. The scTCR will be expressed containing the carboxylterminal biotinylation sequence, BirA, see Schatz, P., Bio/Technology,11, 1138-1143 (1993), as described in aim 2, that can bind free biotinin vivo or in vitro in the presence of biotin ligase, Schatz, P. supra,an enzyme capable of adding a single biotin molecule to the BirA site.Monovalent bispecific molecules can be efficiently formed using thisapproach. An attraction for using this approach is the stronginteraction between avidin and biotin (10⁻¹⁵ M) (see, Green, N., MethodsEnzymol., (1990) 184, 85-133 and the high probability of formingheterodimers (scTCR:scFv) compared to homodimer formation (scTCR:scTCRor scFv:scFv).

Use of additional joining molecules are contemplated. For example,another approach can be used in which it is possible to link a desiredsc-TCR and sc-Fv together by using the fos/jun leucine zipper, see forexample, Patel et al., Proc. Natl. Acad. Sci. USA, (1994) 91, 7360-7364and Segal et al., Annals. New York Academy of Sciences, (1991) 636,288-294. Again, each molecule would be made separately. For example, thescFv will have a carboxyl terminal fos sequence and the scTCR will beengineered to have a carboxyl terminal jun sequence. A single cysteineresidue will flank either side of the fos and jun moieties to facilitatethe formation of disulfide bonds to enhance the stability of theinteraction. Also like the avidin/biotin approach, this technology hasthe advantage of producing heterodimers at a very high frequencycompared to homodimer formation, Patel et al., supra.

The invention has been described with reference to preferred embodimentsthereof, however, it will be appreciated that those skilled in the art,upon consideration of this disclosure, may make modifications andimprovements within the spirit and scope of the invention. All documentsreferenced herein are incorporated by reference.

1-45. (canceled)
 46. A method for preventing or treating a cancer in amammal in which the cancer features pHLA-expressing tumor cells, themethod comprising: a) administering to the mammal a single-chainpolyspecific binding protein comprising at least one single-chain T-Cellreceptor (sc-TCR) and at least one single-chain Fv (sc-Fv), wherein thesc-TCR specifically binds to a HLA complexed with a known peptideantigen on tumor cells and the sc-Fv specifically binds CD3 on thesurface of immune cells, and wherein the sc-TCR comprises a V-β chainand a V-α chain covalently linked by a peptide linker and furthercomprises a C-β chain or fragment thereof fused to the C-terminus of theV-β chain; b) forming a specific binding complex through thesingle-chain polyspecific binding protein interactions with the knownpeptide antigen HLA complex on the tumor cells and with the CD3 moleculeon the immune cells sufficient to activate the immune cells; and c)damaging or killing the tumor cells with the activated immune cellssufficient to prevent or treat the cancer in the mammal.
 47. (canceled)48. The method of claim 46, wherein the peptide linker links theC-terminus of the V-α chain to the N-terminus of the V-β chain.
 49. Themethod of claim 46, wherein the peptide linker comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:
 50. The method of claim 46, wherein the peptide linkerconsists of between 5 and 25 amino acids.
 51. The method of claim 46,wherein the sc-TCR further comprises a TCR C-α chain or fragment thereoffused to the C-terminus of the V-α chain and the N-terminus of thepeptide linker.
 52. The method of claim 46, wherein the sc-TCR and thesc-Fv are adjoined by a peptide linker.
 53. The method of claim 52,wherein the peptide linker adjoining the sc-TCR and the sc-Fv comprisesthe amino acids sequence of SEQ ID NO: 1 or SEQ ID NO:
 4. 54. The methodof claim 46, wherein the sc-Fv is humanized.
 55. The method of claim 46,wherein a C-0 chain is a human C-0 chain.
 56. The method of claim 46,wherein the immune cells are cytotoxic T lymphocytes.
 57. The method ofclaim 46, wherein the tumor cells comprise HLA-A2-peptide antigencomplexes and the peptide antigen is a peptide fragment of the humanwild-type tumor suppressor protein p53 restricted by HLA-A2.
 58. Themethod of claim 46, wherein the tumor cells comprise HLA-A2-peptideantigen complexes and the peptide antigen is a peptide fragment of theHER-2 restricted by HLA-A2.
 59. The method of claim 46, wherein themammal is a human.